MTS2 gene

ABSTRACT

The present invention relates to somatic mutations in the Multiple Tumor Suppressor (MTS) gene in human cancers and their use in the diagnosis and prognosis of human cancer. The invention further relates to germ line mutations in the MTS gene and their use in the diagnosis of predisposition to melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin&#39;s lymphoma, CLL, and cancers of the pancreas, breast, thyroid, ovary, uterus, testis, kidney, stomach and rectum. The invention also relates to the therapy of human cancers which have a mutation in the MTS gene, including gene therapy, protein replacement therapy and protein mimetics. Finally, the invention relates to the screening of drugs for cancer therapy.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation of application Ser. No.08/486,047, filed Jun. 7, 1995, now U.S. Pat. No. 5,994,095, which is acontinuation-in-part of application Serial Nos. PCT/US95/03316, filedMar. 17, 1995, Ser. No. 08/251,938, filed Jun. 1, 1994, now abandoned,Ser. No. 08/215,087, filed Mar. 18, 1994, now abandoned, and Ser. No.08/215,086, filed Mar. 18, 1994, now abandoned, which are allincorporated herein by reference. Application Ser. No. 08/251,938 inturn is a continuation-in-part of application Ser. No. 08/227,369, filedApr. 14, 1994, now abandoned, which is a continuation-in-part ofapplication Ser. No. 08/214,582, filed Mar. 18, 1994, now abandoned,which are all incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to somatic mutations in the Multiple TumorSuppressor (MTS) gene in human cancers and their use in the diagnosisand prognosis of human cancer. The invention further relates to germlinemutations in the MTS gene and their use in the diagnosis ofpredisposition to cancer, such as melanoma, ocular melanoma, leukemia,astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin's lymphoma,multiple myeloma, sarcoma, myosarcoma, cholangiocarcinoma, squamous cellcarcinoma, CLL, and cancers of the pancreas, breast, brain, prostate,bladder, thyroid, ovary, uterus, testis, kidney, stomach, colon andrectum. The invention also relates to the therapy of human cancers whichhave a mutation in the MTS gene, including gene therapy, proteinreplacement therapy and protein mimetics. Finally, the invention relatesto the screening of drugs for cancer therapy.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated herein byreference, and for convenience are referenced in the following text andrespectively grouped in the appended List of References.

The genetics of cancer is complicated, involving multiple dominant,positive regulators of the transformed state (oncogenes) as well asmultiple recessive, negative regulators (tumor suppressor genes). Overone hundred oncogenes have been characterized. Fewer than a dozen tumorsuppressor genes have been identified, but the number is expected toincrease beyond fifty (Knudson, 1993).

The involvement of so many genes underscores the complexity of thegrowth control mechanisms that operate in cells to maintain theintegrity of normal tissue. This complexity is manifested in anotherway. So far, no single gene has been shown to participate in thedevelopment of all, or even the majority of human cancers. The mostcommon oncogenic mutations are in the H-ras gene, found in 10-15% of allsolid tumors (Anderson et al., 1992). The most frequently mutated tumorsuppressor gene is the p53 gene, mutated in roughly 50% of all tumors.Without a target that is common to all transformed cells, the dream of a“magic bullet” that can destroy or revert cancer cells while leavingnormal tissue unharmed is improbable. The hope for a new generation ofspecifically targeted antitumor drugs may rest on the ability toidentify tumor suppressor genes or oncogenes that play general roles incontrol of cell division.

The tumor suppressor genes, which have been cloned and characterized,influence susceptibility to: 1) retinoblastoma (RB1); 2) Wilms' tumor(WT1); 3) Li-Fraumeni (TP53); 4) Familial adenomatous polyposis (APC);5) Neurofibromatosis type 1 (NF1); 6) Neurofibromatosis type 2 (NF2); 7)von Hippel-Lindau syndrome (VHL); and 8) Multiple endocrine neoplasiatype 2A (MEN2A).

Tumor suppressor loci that have been mapped genetically but not yetisolated include genes for: Multiple endocrine neoplasia type 1 (MEN1);Lynch cancer family syndrome 2 (LCFS2); Familial breast cancer (BRCA1);Neuroblastoma (NB); Basal cell nevus syndrome (BCNS); Beckwith-Wiedemannsyndrome (BWS); Renal cell carcinoma (RCC); Tuberous sclerosis 1 (TSC1);and Tuberous sclerosis 2 (TSC2). The tumor suppressor genes that havebeen characterized to date encode products with similarities to avariety of protein types, including DNA binding proteins (WT1),ancillary transcription regulators (RB1), GTPase activating proteins orGAPs (NF1), cytoskeletal components (NF2), membrane bound receptorkinases (MEN2A), and others with no obvious similarity to known proteins(APC and VHL).

In many cases, the tumor suppressor gene originally identified throughgenetic studies has been shown in some sporadic tumors to be lost ormutated. This result suggests that regions of chromosomal aberration maysignify the position of important tumor suppressor genes involved bothin genetic predisposition to cancer and in sporadic cancer.

One of the hallmarks of several tumor suppressor genes characterized todate is that they are deleted at high frequency in certain tumor types.The deletions often involve loss of a single allele, a so-called loss ofheterozygosity (LOH), but may also involve homozygous deletion of bothalleles. For LOH, the remaining allele is presumed to be nonfunctional,either because of a preexisting inherited mutation, or because of asecondary sporadic mutation.

Melanoma is a common cancer afflicting one in every hundred Americans(American Cancer Society, 1992). Environmental influences, such asexposure to ultraviolet light, play a large role in melanoma incidence,but heredity is also a contributing factor. A gene for familialmelanoma, MLM, has been mapped to chromosome 9p21 (Cannon-Albright etal., 1992; Nancarrow et al., 1993; Gruis et al., 1993; Goldstein et al.,1994). Possession of a single predisposing allele at the MLM locusincreases the probability that an individual will develop melanoma by upto approximately 50-fold. MLM belongs to the growing family of suspectedtumor suppressor genes. Predisposition to melanoma is inherited as adominant Mendelian trait, yet predisposing mutations in MLM are thoughtto act as somatic recessive alleles in the manner originally proposed byKnudson (1971). In a predisposed individual who carries one wild-typeand one mutant MLM allele, dividing cells undergo secondary mutationalevents that involve loss or inactivation of the wild-type copy of MLM,thereby uncovering the inherited mutant MLM allele. Conversely, a singlewild-type copy of the gene prevents the onset of malignancy.

Chromosomal aberrations in the vicinity of MLM at 9p21 have beenextensively characterized in several different tumor types, includingglioma cell lines, non-small cell lung lines and acute lymphoblasticleukemia lines (Olopade et al., 1992; Olopade et al., 1993; Lukeis etal., 1990; Diaz et al., 1988; Middleton et al., 1991; Fountain et al.,1992; Cheng et al., 1993; James et al., 1993). Thus, based on thefrequency of 9p21 chromosomal abnormalities in non-melanoma tumor cells,it is probable the MLM region contains a gene (or genes) thatparticipates at least in the progression of several different tumortypes. These events involve LOH as well as a high frequency ofhomozygous deletion.

Cells in tissues have only three serious options in life—they can growand divide, not grow but stay alive, or die by apoptosis. Tumors mayarise either by inappropriate growth and division or by cells failing todie when they should. One of the mechanisms for controlling tumor growthmight involve direct regulation of the cell cycle. For example, genesthat control the decision to initiate DNA replication are attractivecandidates for oncogenes or tumor suppressor genes, depending on whetherthey have a stimulatory or inhibitory role in the process. Progressionof eukaryotic cells through the cell cycle (G₁, S, G₂ and M phases) isgoverned by the sequential formation, activation and subsequentinactivation of a series of cyclin/cyclin-dependent kinase (Cdk)complexes. Cyclin D's/Cdk2,4,5, Cyclin E/Cdk2, Cyclin A/Cdk2 and CyclinB/A/Cdk2 have been shown to be involved in this process. Cyclin D's andCdk2, Cdk4 and Cdk5 have been implicated in the transition from G₁ to S;that is, when cells grow and decide whether to begin DNA replication.Additional cell cycle control elements have recently been discovered.These elements are inhibitors of Cdks (Cdk inhibitors, CkI), and includeFar1, p21, p40, p20 and p16. (Marx, 1994; Nasmyth & Hunt, 1993).

Recently, several oncogenes and tumor suppressor genes have been foundto participate directly in the cell cycle. For example, one of thecyclins (proteins that promote DNA replication) has been implicated asan oncogene (Motokura et al., 1991; Lammie et al., 1991; Withers et al.,1991; Rosenberg et al., 1991), and tumor suppressor Rb interacts withthe primary cyclin-binding partners, the Cdks (Ewen et al., 1993).Identification of a melanoma susceptibility locus would open the way forgenetic screening of individuals to assess, for example, the increasedrisk of cancer due to sunlight exposure. The MTS may also predispose toa large number of other cancer sites, including but not limited to,leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin'slymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiocarcinoma,squamous cell carcinoma, CLL, and cancers of the pancreas, breast,brain, prostate, bladder, thyroid, ovary, uterus, testis, kidney,stomach, colon and rectum. In addition, since MTS influences progressionof several different tumor types, it should be useful for determiningprognosis in cancer patients. Thus, MTS may serve as the basis fordevelopment of very important diagnostic tests, one capable ofpredicting the predisposition to cancer, such as melanoma, ocularmelanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma,Hodgkin's lymphoma, multiple myeloma, sarcoma, myosarcoma,cholangiocarcinoma, squamous cell carcinoma, CLL, and cancers of thepancreas, breast, brain, prostate, bladder, thyroid, ovary, uterus,testis, kidney, stomach, colon and rectum, and one capable of predictingthe prognosis of cancer. Furthermore, since MTS is involved in theprogression of multiple tumor types, MTS may provide the means, eitherdirectly or indirectly, for a general anti-cancer therapy by virtue ofits ability to suppress tumor growth. For example, restoration of thenormal MTS function to a tumor cell may transmute the cell intonon-malignancy.

SUMMARY OF THE INVENTION

The present invention relates to somatic mutations in the Multiple TumorSuppressor (MTS) gene in human cancers and their use in the diagnosisand prognosis of human cancer. The invention further relates to germlinemutations in the MTS gene and their use in the diagnosis ofpredisposition to many cancers, such as melanoma, ocular melanoma,leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin'slymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiocarcinoma,squamous cell carcinoma, CLL, and cancers of the pancreas, breast,brain, prostate, bladder, thyroid, ovary, uterus, testis, kidney,stomach, colon and rectum. The invention also relates to the therapy ofhuman cancers which have a mutation in the MTS gene, including genetherapy, protein replacement therapy and protein mimetics. Finally, theinvention relates to the screening of drugs for cancer therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows Kindred 3137. All melanoma cases carry the susceptiblehaplotype. Other cancers in individuals carrying the susceptiblehaplotypes are also shown. The legends are as follows: filled circle orsquare indicates melanoma; partially filled circle or square indicatesother cancer; “/” indicates deceased; “*” indicates that individual isunknown with regard to susceptible haplotype; “**” indicates thatindividual appears to carry susceptible haplotype.

FIG. 1B shows Kindred 3161. All melanoma cases carry the susceptiblehaplotype. Other cancers have not been haplotyped. The legends are asfollows: filled circle or square indicates melanoma; partially filledcircle or square indicates other cancer; “/” indicates deceased; “=”indicates appears elsewhere in kindred; “3” in pentagon indicatesmultiple marriage.

FIG. 1C shows Kindred 3355. All melanoma cases carry the susceptiblehaplotype. Other cancers have not been haplotyped. The legends are asfollows: filled circle or square indicates melanoma; partially filledcircle or square indicates other cancer; “/” indicates deceased.

FIG. 1D shows Kindred 1771 and the occurrence of melanoma and othercancers. A mutation was identified in MTS in this kindred. The legendsare as follows: an “*” indicates a confirmed mutation carrier; filledcircle or square indicates melanoma; partially filled circle or squareindicates other cancer (colon in this kindred); “/” indicates deceased.

FIG. 2 shows YAC and P1 clones in the region bounded by IFNA-s andD9S171. The centromere is to the right. For P1 clones, the arrow pointsin the direction of the T7 promoter sequence in the vector. YACs thatare grouped together represent clones that are similar based on mappingSTSs in the region. These YACs are presumed not to be identical. YACsA5, B11, C6 and F9 contain IFN-1 and IFN-s. YACs D1, F5 and E3 containD9S126 and D9S171. Neither the proximal ends of YACs that include D9S171nor the distal ends of YACs that include IFNA-s are shown. Distances arenot necessarily drawn to scale. The markers internal to IFNA-s andD9S171 are depicted in FIG. 2. Markers that begin with “c” are derivedfrom cosmid end sequences. The cosmids are not shown. The distancesbetween c1.b and c5.3 and between 760-L and D9S171 are unknown.

FIG. 3 shows a diagram of deletions observed in melanoma cell lines. Thedeletions fall into 12 classes, based on the set of markers which aredeleted. Eleven cell lines lacked all markers depicted in the figure.This class is not shown. The number of representatives of each of the 12other classes is shown in the column labeled “# lines.” Locations of thedeletion breakpoints for classes 1-10 are portrayed as falling at themarker adjacent to the deleted DNA; that is, the last positive marker inthe series leading up to the deletion. For classes 11 and 12, the sitesof deletions are shown by filled triangles.

FIG. 4A shows a map of cosmid c5. Relevant STSs used for the deletionanalysis are shown, as are cosmids and P1s. The c1.b marker liesproximal to P1-1062 and is not shown. The transcriptional orientationsof MTS1 and MTS2 are shown by arrows.

FIG. 4B shows a restriction map and STS map of cosmid c5. Positions ofcoding exons for MTS1 and MTS2 are shown as thick bars. “E1” and “E2”mean “coding exon 1” and “coding exon 2,” respectively. “B” is BamHI,“S” is SaII, “R1’ is EcoRI and “R5” is EcoRV.

FIGS. 5A and 5B show a comparison of the genomic sequence containing a5′ untranslated region, exon 1, and part of intron 1 for MTS1 with thepublished sequence for p16 (Serrano et al., 1993). The start codon(underlined) is located at position 867 and a splice site (arrow) atposition 1016. The MTS1 sequence shown in FIGS. 5A-B is SEQ ID NO:3. Thep16 sequence shown in FIG. 5B is SEQ ID NO:24.

FIGS. 6A and 6B show a comparison of the genomic sequence containingpart of intron 1, exon 2 and part of intron 2 for MTS1, with thepublished sequence for p16 (Serrano et al., 1993). Splice sites (arrows)are located before position 192 and after position 498. The MTS1sequence shown in FIGS. 6A-B is SEQ ID NO:4. The p16 sequence shown inFIG. 6A is identical to nucleotides 192-498 of SEQ ID NO:4.

FIGS. 7A and 7B show a comparison of the genomic sequence containingpart of intron 1, “exon 2,” and follow-through sequences for MTS2 withthe published p16 sequence. The “Exon 2” sequence is similar to exon 2of MTS1 from nucleotides 273 to 580. The splice site in MTS2, and thosein p16, are shown by arrows. The point where divergence begins isindicated by °. The termination codon for MTS2 is present in exon 2 atposition 532 and is indicated by an “*”. The MTS2 sequence shown inFIGS. 7A-B is SEQ ID NO:5. The p16 sequence shown in FIG. 7A isidentical to nucleotides 192-498 of SEQ ID NO:4.

FIG. 8 shows a comparison of the MTS1 and MTS2 DNA sequences includingexon 2 and part of each surrounding intron. The positions of the 3′splice junction of intron 1 and the 5′ splice junction of intron 2 forMTS1 are shown by triangles. The divergence point near the 3′ end ofcoding exon 2 is indicated by an arrow. The MTS1 sequence showncorresponds to nucleotides 92-548 of SEQ ID NO:4. The MTS2 sequenceshown corresponds to nucleotides 174-630 of SEQ ID NO:5.

FIG. 9 shows deletions in tumor cell lines of various STSs. Positivecontrols and negative controls were included in every PCR experiment andcell lines in which only one or two of the STSs were deleted (e.g.,class 21) were retested at least twice.

FIGS. 10A-C show expression of MTS2 mRNA. FIG. 10A shows the relativelevel of MTS2 transcript in RNA (Clonetech) derived from various humantissues: lane 1-brain; lane 2-breast; lane 3-kidney; lane 4-lung; lane5-lymphocyte; lane 6-ovary; lane 7-pancreas; lane 8-prostate; lane9-spleen; lane 10-stomach; lane 11-thymus. The origins of products withdifferent than expected molecular weights (see lane 1) are unknown. FIG.10B shows the relative MTS2 transcript level in human lymphocytes as afunction of time after mitogenic induction: lane 1-0 hours; lane 2-1hour; lane 3-2 hours; lane 4-4 hours; lane 5-8 hours; lane 6-16 hours;lane 7-24 hours; lane 8-32 hours; lane 9-40 hours; lane 10-48 hours;lane 11-56 hours; lane 12-64 hours. A majority of the cells were in Sphase 40-50 hours after induction. FIG. 10C shows MTS2 transcript levelas a function of Rb status. The Rb⁻ cell lines are: Lane 1-WERI; lane2-CaSki; lane 3-SiHa; lane 4-C33A; lane 5-5637; lane 6-MDA MB 468. TheRb⁺ cell lines are: lane 7-T24; lane 8-HaCaT; lane 9-ZR75; lane10-Bristol 8; lane 11-UMSCC2; lane 12-diploid human fibroblast MRC5,passage 28; lane 13-KIT (Hori et al., 1987).

FIG. 11 shows the cDNA sequence (and the encoded polypeptide) for MTS2including 5′-untranslated region. The beginning of exon 2 is located atposition 491 and is indicated by an arrow. The cDNA sequence is shown asSEQ ID NO:15 and the amino acid sequence is shown as SEQ ID NO: 16.

FIGS. 12A and 12B show the cDNA sequence (and the encoded polypeptide)of MTS1E1β. Splice sites are indicated by arrows. Exon 2 begins atposition 335 and exon 3 begins at position 642. The cDNA sequence isshown as SEQ ID NO:13 and the amino acid sequence is shown by SEQ IDNO:14.

FIG. 13 is a physical map of the P16 region. The positions of exon 1α(E1α), exon 1β (E1β), exon 2 (E2) and exon 3 (E3) are indicated by thefilled boxes. The positions of restriction sites Eco R1 (R1), Eco RV(RV), and Sal I (S) are indicated. Above the restriction map are genomicclones cosmid c5 and P1 1063. Below the map are the deletions in celllines A375 and SK-mel 93. The dashed line represents deleted DNA. Theexact location of the distal breakpoint is not known in either A375 orSK-mel 93. However, these have been mapped to the interval between E1αand the STS c5.3 (Kamb et al., 1994b; Stone et al., unpublished).

FIG. 14 shows the alignment between mouse and human P16 β transcriptsequences. Capital letters indicate identical nucleotides. The stopcodons in the p16 reading frame are underlined. The splice junctionbetween E1β and E2 is indicated with a caret (v). The mouse β sequenceis shown as SEQ ID NO:25. The human β sequence is identical tonucleotides 193-461 of SEQ ID NO:13.

FIG. 15 shows the expression of the a transcript in cell lines thatcontain deletions of E1β. cDNA was derived from total RNA isolated fromthe indicated samples. A radio-labeled primer was included in thereactions to amplify the P16 transcripts. Equal volumes of the α and βamplifications were mixed, and the products were resolved on adenaturing 5% polyacrylamide gel: lane 1-quiescent T cells; lane 2-cellline SK-mel 93; lane 3-cell line A375.

FIGS. 16A-D show the expression of P16 transcripts. A radio-labeledprimer was included in the reactions to amplify the P16 transcripts andthe products were resolved on a denaturing 5% polyacrylamide gel. InFIGS. 16A and 16D the α and β reactions from a common sample were mixedprior to electrophoresis. FIG. 16A shows the relative levels of P16transcripts in RNA derived from various human tissues: lane 1, brain;lane 2, breast; lane 3, kidney; lane 4, lung; lane 5, lymphocyte; lane6, ovary; lane 7, pancreas; lane 8, prostate; lane 9, spleen; lane 10,stomach; lane 11, thymus. FIG. 16B shows the relative amount of the βtranscript in human lymphocytes as a function of time after mitogenicinduction: lane 1, 0 hours; lane 2, 1 hour; lane 3, 2 hours; lane 4, 4hours; lane 5, 8 hours; lane 6, 16 hours; lane 7, 24 hours; lane 8, 32hours; lane 9, 40 hours; lane 10, 48 hours; lane 11, 56 hours; lane 12,64 hours. FIG. 16C shows the relative amount of the α transcript inhuman lymphocytes as a function of time after mitogenic induction:Lanes, same as in FIG. 16B, but the 1 hour time point was omitted. Theexpression of other molecules that are either suspected to influencecell-cycle progression or that are regulated at the transcriptionallevel during the cell-cycle was also analyzed. In agreement withprevious results, levels of CDK4 and GoS 2 (a molecule of unknownfunction, but whose transcription is induced when quiescent T cellsenter the cell cycle) increased upon T cell induction (Russell andForsdyke, 1991; Matsushime et al., 1992; Geng and Weinberg, 1993). Incontrast, the RNA levels of p27 appeared unchanged during the course ofthe experiment (Toyoshima and Hunter, 1994; Kato et al., 1994). FIG. 16Dshows P16 transcripts as a function of Rb status. Rb⁻ cell lines: lane1, WERI; lane 2, CaSki; lane 3, SiHa; lane 4, C33A; lane 5, 5637; lane6, MDA MB 468. Rb⁺ cell lines: lane 7, T24; lane 8, HaCaT; lane 9, Zr75;lane 10, Bristol 8; lane 11, UMSCC2; lane 12, diploid human fibroblastMRC5, passage 28; lane 13, KIT (Hori et al., 1987).

FIG. 17 shows the cDNA sequence for MTS 1 including noncoding portionsof the cDNA. The triangles indicate splice junctions. The dashes in thesequence at the second splice junction only emphasize this splicejunction, they do not indicate missing bases. This sequence is SEQ IDNO:36.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to somatic mutations in the Multiple TumorSuppressor (MTS) gene in human cancers and their use in the diagnosisand prognosis of human cancer. The invention further relates to germline mutations in the MTS gene and their use in the diagnosis ofpredisposition to various cancers, such as melanoma, ocular melanoma,leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin'slymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiocarcinoma,squamous cell carcinoma, CLL, and cancers of the pancreas, breast,brain, prostate, bladder, thyroid, ovary, uterus, testis, kidney,stomach, colon and rectum. The invention also relates to the therapy ofhuman cancers which have a mutation in the MTS gene, including genetherapy, protein replacement therapy and protein mimetics. Finally, theinvention relates to the screening of drugs for cancer therapy.

The present invention provides an isolated polynucleotide comprisingall, or a portion of the MTS locus or of a mutated MTS locus, preferablyat least eight bases and not more than about 100 Kb in length. Suchpolynucleotides may be antisense polynucleotides. The present inventionalso provides a recombinant construct comprising such an isolatedpolynucleotide, for example, a recombinant construct suitable forexpression in a transformed host cell.

Also provided by the present invention are methods of detecting apolynucleotide comprising a portion of the MTS locus or its expressionproduct in an analyte. Such method may further comprise the step ofamplifying the portion of the MTS locus, and may further include a stepof providing a set of polynucleotides which are primers foramplification of said portion of the MTS locus. The method is useful foreither diagnosis of the predisposition to cancer or the diagnosis orprognosis of cancer.

The present invention also provides isolated antibodies, preferablymonoclonal antibodies, which specifically bind to an isolatedpolypeptide comprised of at least five amino acid residues encoded bythe MTS locus.

The present invention also provides kits for detecting in an analyte apolynucleotide comprising a portion of the MTS locus, the kitscomprising a polynucleotide complementary to the portion of the MTSlocus packaged in a suitable container, and instructions for its use.

The present invention further provides methods of preparing apolynucleotide comprising polymerizing nucleotides to yield a sequencecomprised of at least eight consecutive nucleotides of the MTS locus;and methods of preparing a polypeptide comprising polymerizing aminoacids to yield a sequence comprising at least five amino acids encodedwithin the MTS locus.

In addition, the present invention provides methods of screening drugsfor cancer therapy to identify suitable drugs for restoring MTS geneproduct function.

Finally, the present invention provides the means necessary forproduction of gene-based therapies directed at cancer cells. Thesetherapeutic agents may take the form of polynucleotides comprising allor a portion of the MTS locus placed in appropriate vectors or deliveredto target cells in more direct ways such that the function of the MTSprotein is reconstituted. Therapeutic agents may also take the form ofpolypeptides based on either a portion of, or the entire proteinsequence of MTS. These may functionally replace the activity of MTS invivo.

It is a discovery of the present invention that the MTS locus (referredto in the prior art as Melanoma (MLM) locus), which predisposesindividuals to melanoma and other cancers, is a gene encoding MTS1,which has been found to be an inhibitor of Cdks, particularly Cdk4*.This gene is termed MTS1 herein. It is also a discovery of the presentinvention that the MTS locus contains a second coding sequence, termedMTS2, which is very similar to MTS1 over part of its sequence. It isalso a discovery of the present invention that the MTS1 gene has twoseparate promoters—α and β. When the α promoter is used the resultingmRNA is composed of exon 1α, exon 2 and exon 3. This is referred to asMTS1. When the β promoter is used the resulting mRNA is composed of exon1β, exon 2 and exon 3. This is referred to as MTS1E1β. It is a discoveryof the present invention that mutations in the MTS locus in the germlineare indicative of a predisposition to melanoma and to other cancers.Finally, it is a discovery of the present invention that somaticmutations in the MTS locus are associated with most, if not all tumortypes, and thus represent a general indicator of cancer or of prognosisof cancer. The mutational events of the MTS locus can involve deletions,insertions and point mutations within the coding sequence and thenon-coding sequence.

The MLM locus was first located genetically by showing dramatic linkagein several Utah kindreds and one Texas kindred between genetic markersand melanoma predisposition (Cannon-Albright, 1992). The region definedby recombinants in the kindreds is flanked by D9S736 and D9S171.Subsequently, these and other genetic markers were used to localize thegene by analysis of homozygous deletions in both melanoma andnon-melanoma tumor cell lines containing deletions. The minimum area ofoverlap of the deletions was flanked by IFNA-s and D9S171. YAC librarieswere screened to identify genomic clones surrounding these markers. P1clones were isolated as part of a chromosomal walk and were contiguousfrom IFNA-s to D9S171 except for two gaps. Specific sequence-taggedsites (“STS”) were prepared to construct a more detailed molecular map.Using these markers and a deletion analysis, a region of deletionoverlap centered around markers c5.1 and c5.3, markers found on cosmid 5(c5). The most frequently deleted marker was c5.3, which thus was veryclose to MTS.

An analysis of c5 for the presence of “CpG” islands showed that itcontained at least one candidate gene for MTS. DNA sequences of EcoRIfragments of c5 were determined and compared against sequences fromGenBank. Two distinct regions of c5 were identified that were similar toa region of a previously identified gene encoding human Cdk4 inhibitor,or p16 (Serrano et al., 1993). These two candidate genes are called MTS1and MTS2. Screening cDNA libraries of lymphocyte, fetal brain and normalbreast with a probe from Exon 2 of MTS1 identified an additionalcandidate called MTS1E1β.

A detailed comparison of the genomic sequence from c5 with the p16 mRNAsequence revealed that MTS1 contained a stretch of 307 bp that wasidentical to a portion of the p16 coding sequence. This stretch ofnucleotides in MTS1 was flanked by recognizable splice junctionsequences. Further characterization of MTS1 showed that it included theentire coding sequence of p16 plus two introns. Intron 1 was located 126bp downstream from the translational start site; Intron 2 was located 11bp upstream from the translational stop site. The two introns dividedthe coding sequence of p16 into three regions, a 5′ region of 126 bp(coding Exon 1), a middle region of 307 bp (coding Exon 2), and a 3′region of 11 bp (coding Exon 3).

MTS2 contained a region of DNA sequences nearly identical to p16 thatextended from the 5′ end of coding Exon 2 roughly 200 bp toward intron2. However, the sequence similarity decreased to a point 51 bp upstreamof Intron 2 in MTS1, where the two sequences diverged completely. Thiscorresponds to the location of the final codon of MTS2. Comparison ofsequences from MTS1 and MTS2 showed that the sequence similarity betweenthese two genes also extended nearly 50 nucleotides upstream from the 3′splice junction of intron 1. Thus, portions of noncoding DNA were moreconserved than some areas of presumptive coding DNA. To exclude thepossibility that the sequence divergence in coding DNA might be acloning artifact, PCR primers were designed to amplify specificallyacross the sequence divergence point of MTS2. These primers amplified afragment of the predicted size from cosmid P1 and genomic DNA.Therefore, the divergent sequence located near the 3′ end of Exon 2 inMTS2 is a bona fide genomic sequence.

MTS1E1β contains an Exon 1, called Exon 1β or E1β, which has a differentsequence than found in Exon 1 of MTS1 and MTS2. MTS1E1β also containsExon 2 (E2) and Exon 3 (E3) which are identical to Exons 2 and 3 ofMTS1. Exon 1β is located upstream of Exon 1 of MTS1 and does not containany coding sequence. As a result MTS1E1β encodes a p10 which has atranslation start site at the first ATG of Exon 2.

MTS1 and MTS2 were tested for correspondence with the geneticsusceptibility locus MTS by analyzing genomic DNA, using Exon 2, fromindividuals presumed to carry MLM predisposing alleles. DNApolymorphisms were identified in Exon 2 of MTS1 in one of eightindividuals. The mutation was a single nucleotide substitution,resulting in an amino acid change. This polymorphism segregated with theMLM predisposing allele.

The preponderance of lesions in MTS1 (deletions and nucleotidesubstitutions) indicates that MTS1 or a closely linked locus contributesto the tumor phenotype. Cells that suffer these lesions enjoy aselective advantage over cells that do not. The alternative explanation,that the lesions are random events having nothing to do with cellgrowth, is unlikely for several reasons. First, the high correlationbetween tumor phenotype and mutation at MTS1 implies a causal relationbetween MTS1 mutations and tumor formation. Second, MTS1 influencessusceptibility to melanoma, and thus is implicated independently as atumor suppressor gene. Third, the biochemical function of p16 as apotent inhibitor of a Cdk neatly fits a model where MTS1 acts in vivo asa general inhibitor of the onset of DNA replication.

According to the diagnostic and prognostic method of the presentinvention, alteration of the wild-type MTS locus is detected. Inaddition, the method can be performed by detecting the wild-type MTSlocus and confirming the lack of a predisposition or neoplasia.“Alteration of a wild-type gene” encompasses all forms of mutationsincluding deletions, insertions and point mutations in the coding andnoncoding regions. Deletions may be of the entire gene or only a portionof the gene. Point mutations may result in stop codons, frameshiftmutations or amino acid substitutions. Somatic mutations are those whichoccur only in certain tissues, e.g., in the tumor tissue, and are notinherited in the germline. Germline mutations can be found in any of abody's tissues and are inherited. If only a single allele is somaticallymutated, an early neoplastic state is indicated. However, if bothalleles are mutated, then a late neoplastic state is indicated. Thefinding of MTS mutations thus provides both diagnostic and prognosticinformation. An MTS allele which is not deleted (e.g., that found on thesister chromosome to a chromosome carrying an MTS deletion) can bescreened for other mutations, such as insertions, small deletions, andpoint mutations. It is believed that many mutations found in tumortissues will be those leading to decreased expression of the MTS geneproduct. However, mutations leading to non-functional gene productswould also lead to a cancerous state. Point mutational events may occurin regulatory regions, such as in the promoter of the gene, leading toloss or diminution of expression of the mRNA. Point mutations may alsoabolish proper RNA processing, leading to loss of expression of the MTSgene product, or a decrease in mRNA stability or translation efficiency.

Useful diagnostic techniques include, but are not limited to fluorescentin situ hybridization (FISH), direct DNA sequencing, PFGE analysis,Southern blot analysis, single stranded conformation analysis (SSCA),RNase protection assay, allele-specific oligonucleotide (ASO), dot blotanalysis and PCR-SSCP, as discussed in detail further below.

Predisposition to cancers, such as melanoma and the other cancersidentified herein, can be ascertained by testing any tissue of a humanfor mutations of the MTS gene. For example, a person who has inherited agermline MTS mutation would be prone to develop cancers. This can bedetermined by testing DNA from any tissue of the person's body. Mostsimply, blood can be drawn and DNA extracted from the cells of theblood. In addition, prenatal diagnosis can be accomplished by testingfetal cells, placental cells or amniotic fluid for mutations of the MTSgene. Alteration of a wild-type MTS allele, whether, for example, bypoint mutation or by deletion, can be detected by any of the meansdiscussed herein.

In order to detect the alteration of the wild-type MTS gene in a tissue,it is helpful to isolate the tissue free from surrounding normaltissues. Means for enriching a tissue preparation for tumor cells areknown in the art. For example, the tissue may be isolated from paraffinor cryostat sections. Cancer cells may also be separated from normalcells by flow cytometry. These techniques, as well as other techniquesfor separating tumor cells from normal cells, are well known in the art.If the tumor tissue is highly contaminated with normal cells, detectionof mutations is more difficult.

A rapid preliminary analysis to detect polymorphisms in DNA sequencescan be performed by looking at a series of Southern blots of DNA cutwith one or more restriction enzymes, preferably a large number ofrestriction enzymes. Each blot contains a series of normal individualsand a series of cancer cases, tumors, or both. Southern blots displayinghybridizing fragments (differing in length from control DNA when probedwith sequences near or including the MTS locus) indicate a possiblemutation. If restriction enzymes which produce very large restrictionfragments are used, then pulsed field gel electrophoresis (“PFGE”) isemployed.

Detection of point mutations may be accomplished by molecular cloning ofthe MTS allele(s) and sequencing that allele(s) using techniques wellknown in the art. Alternatively, the gene sequences can be amplified,using known techniques, directly from a genomic DNA preparation from thetumor tissue. The DNA sequence of the amplified sequences can then bedetermined.

There are six well known methods for a more complete, yet stillindirect, test for confirming the presence of a susceptibilityallele: 1) single stranded conformation analysis (“SSCA”) (Orita et al.,1989); 2) denaturing gradient gel electrophoresis (“DGGE”) (Wartell etal., 1990; Sheffield et al., 1989); 3) RNase protection assays(Finkelstein et al., 1990; Kinszler et al., 1991); 4) allele-specificoligonucleotides (“ASOs”) (Conner et al., 1983); 5) the use of proteinswhich recognize nucleotide mismatches, such as the E. coli mutS protein(Modrich, 1991); and, 6) allele-specific PCR (Rano & Kidd, 1989). Forallele-specific PCR, primers are used which hybridize at their 3′ endsto a particular MTS mutation. If the particular MTS mutation is notpresent, an amplification product is not observed. AmplificationRefractory Mutation System (ARMS) can also be used, as disclosed inEuropean Patent Application Publication No. 0332435 and in Newton etal., 1989. Insertions and deletions of genes can also be detected bycloning, sequencing and amplification. In addition, restriction fragmentlength polymorphism (RFLP) probes for the gene or surrounding markergenes can be used to score alteration of an allele or an insertion in apolymorphic fragment. Such a method is particularly useful for screeningrelatives of an affected individual for the presence of the MTS mutationfound in that individual. Other techniques for detecting insertions anddeletions as known in the art can be used.

In the first three methods (i.e., SSCA, DGGE and RNase protectionassay), a new electrophoretic band appears. SSCA detects a band whichmigrates differentially because the sequence change causes a differencein single-strand, intramolecular base pairing. RNase protection involvescleavage of the mutant polynucleotide into two or more smallerfragments. DGGE detects differences in migration rates of mutantsequences compared to wild-type sequences, using a denaturing gradientgel. In an allele-specific oligonucleotide assay, an oligonucleotide isdesigned which detects a specific sequence, and the assay is performedby detecting the presence or absence of a hybridization signal. In themutS assay, the protein binds only to sequences that contain anucleotide mismatch in a heteroduplex between mutant and wild-typesequences.

Mismatches, according to the present invention, are hybridized nucleicacid duplexes in which the two strands are not 100% complementary. Lackof total homology may be due to deletions, insertions, inversions orsubstitutions. Mismatch detection can be used to detect point mutationsin the gene or its mRNA product. While these techniques are lesssensitive than sequencing, they are simpler to perform on a large numberof tumor samples. An example of a mismatch cleavage technique is theRNase protection method. In the practice of the present invention, themethod involves the use of a labeled riboprobe which is complementary tothe human wild-type MTS gene coding sequence. The riboprobe and eithermRNA or DNA isolated from the tumor tissue are annealed (hybridized)together and subsequently digested with the enzyme RNase A which is ableto detect some mismatches in a duplex RNA structure. If a mismatch isdetected by RNase A, it cleaves at the site of the mismatch. Thus, whenthe annealed RNA preparation is separated on an electrophoretic gelmatrix, if a mismatch has been detected and cleaved by RNase A, an RNAproduct will be seen which is smaller than the full length duplex RNAfor the riboprobe and the mRNA or DNA. The riboprobe need not be thefull length of the MTS mRNA or gene but can be a segment of either. Ifthe riboprobe comprises only a segment of the MTS mRNA or gene, it willbe desirable to use a number of these probes to screen the whole mRNAsequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, throughenzymatic or chemical cleavage. See, e.g., Cotton et al., 1988; Shenk etal., 1975; Novack et al., 1986. Alternatively, mismatches can bedetected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, 1988. Witheither riboprobes or DNA probes, the cellular mRNA or DNA which mightcontain a mutation can be amplified using PCR (see below) beforehybridization. Changes in DNA of the MTS gene can also be detected usingSouthern hybridization, especially if the changes are grossrearrangements, such as deletions and insertions.

DNA sequences of the MTS gene which have been amplified by use of PCRmay also be screened using allele-specific probes. These probes arenucleic acid oligomers, each of which contains a region of the MTS genesequence harboring a known mutation. For example, one oligomer may beabout 30 nucleotides in length, corresponding to a portion of the MTSgene sequence. By use of a battery of such allele-specific probes, PCRamplification products can be screened to identify the presence of apreviously identified mutation in the MTS gene. Hybridization ofallele-specific probes with amplified MTS sequences can be performed,for example, on a nylon filter. Hybridization to a particular probeunder stringent hybridization conditions indicates the presence of thesame mutation in the tumor tissue as in the allele-specific probe.

The most definitive test for mutations in a candidate locus is todirectly compare genomic MTS sequences from cancer patients with thosefrom a control population. Alternatively, one could sequence messengerRNA after amplification, e.g., by PCR, thereby eliminating the necessityof determining the exon structure of the candidate gene.

Mutations from cancer patients falling outside the coding region of MTScan be detected by examining the non-coding regions, such as introns andregulatory sequences near or within the MTS gene. An early indicationthat mutations in noncoding regions are important may come from Northernblot experiments that reveal messenger RNA molecules of abnormal size orabundance in cancer patients as compared to control individuals.

Alteration of MTS mRNA expression can be detected by any techniquesknown in the art. These include Northern blot analysis, PCRamplification and RNase protection. Diminished mRNA expression indicatesan alteration of the wild-type MTS gene. Alteration of wild-type MTSgenes can also be detected by screening for alteration of wild-type MTSprotein. For example, monoclonal antibodies immunoreactive with MTS canbe used to screen a tissue. Lack of cognate antigen would indicate anMTS mutation. Antibodies specific for products of mutant alleles couldalso be used to detect mutant MTS gene product. Such immunologicalassays can be done in any convenient formats known in the art. Theseinclude Western blots, immunohistochemical assays and ELISA assays. Anymeans for detecting an altered MTS protein can be used to detectalteration of wild-type MTS genes. Functional assays, such as proteinbinding determinations, can be used. For example, it is known that MTSprotein binds to Cdks, especially Cdk4. Thus, an assay for the abilityto bind to wild-type MTS protein or Cdk4 can be employed. In addition,assays can be used which detect MTS biochemical function, the inhibitionof Cdks, such as Cdk4, and regulation of the cell cycle. Finding amutant MTS gene product indicates alteration of a wild-type MTS gene.

Mutant MTS genes or gene products can also be detected in other humanbody samples, such as serum, stool, urine and sputum. The sametechniques discussed above for detection of mutant MTS genes or geneproducts in tissues can be applied to other body samples. Cancer cellsare sloughed off from tumors and appear in such body samples. Inaddition, the MTS gene product itself may be secreted into theextracellular space and found in these body samples even in the absenceof cancer cells. By screening such body samples, a simple earlydiagnosis can be achieved for many types of cancers. In addition, theprogress of chemotherapy or radiotherapy can be monitored more easily bytesting such body samples for mutant MTS genes or gene products.

The methods of diagnosis of the present invention are applicable to anytumor in which MTS has a role in tumorigenesis. Deletions of chromosomearm 9p or somatic mutations within the MTS region have been observed inalmost all tumors examined. The diagnostic method of the presentinvention is useful for clinicians, so they can decide upon anappropriate course of treatment.

The primer pairs of the present invention are useful for determinationof the nucleotide sequence of a particular MTS allele using the PCR. Thepairs of single-stranded DNA primers can be annealed to sequences withinor surrounding the MTS gene on chromosome 9p in order to primeamplifying DNA synthesis of the MTS gene itself. A complete set of theseprimers allows synthesis of all of the nucleotides of the MTS genecoding sequences, i.e., the exons. The set of primers preferably allowssynthesis of both intron and exon sequences. Allele-specific primers canalso be used. Such primers anneal only to particular MTS mutant alleles,and thus will only amplify a product in the presence of the mutantallele as a template.

In order to facilitate subsequent cloning of amplified sequences,primers may have restriction enzyme site sequences appended to their 5′ends. Thus, all nucleotides of the primers are derived from MTSsequences or sequences adjacent to MTS, except for the few nucleotidesnecessary to form a restriction enzyme site. Such enzymes and sites arewell known in the art. The primers themselves can be synthesized usingtechniques which are well known in the art. Generally, the primers canbe made using oligonucleotide synthesizing machines which arecommercially available. Given the sequence of the MTS open readingframes shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:13, SEQ ID NO:15 and SEQ ID NO:36, design of particular primers iswell within the skill of the art.

The nucleic acid probes provided by the present invention are useful fora number of purposes. They can be used in Southern hybridization togenomic DNA and in the RNase protection method for detecting pointmutations already discussed above. The probes can be used to detect PCRamplification products. They may also be used to detect mismatches withthe MTS gene or mRNA using other techniques.

Definitions

The present invention employs the following definitions:

“Amplification of Polynucleotides” utilizes methods such as thepolymerase chain reaction (PCR), ligation amplification (or ligase chainreaction, LCR) and amplification methods based on the use of Q-betareplicase. These methods are well known and widely practiced in the art.See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al., 1990(for PCR); and Wu et al., 1989a (for LCR). Reagents and hardware forconducting PCR are commercially available. Primers useful to amplifysequences from the MTS region are preferably complementary to, andhybridize specifically to sequences in the MTS region or in regions thatflank a target region therein. MTS sequences generated by amplificationmay be sequenced directly. Alternatively, but less desirably, theamplified sequence(s) may be cloned prior to sequence analysis. A methodfor the direct cloning and sequence analysis of enzymatically amplifiedgenomic segments has been described by Scharf, 1986.

“Analyte polynucleotide” and “analyte strand” refer to a single- ordouble-stranded polynucleotide which is suspected of containing a targetsequence, and which may be present in a variety of types of samples,including biological samples.

“Antibodies.” The present invention also provides polyclonal and/ormonoclonal antibodies and fragments thereof, and immunologic bindingequivalents thereof, which are capable of specifically binding to theMTS polypeptides and fragments thereof or to polynucleotide sequencesfrom the MTS region, particularly from the MTS locus or a portionthereof. The term “antibody” is used both to refer to a homogeneousmolecular entity, or a mixture such as a serum product made up of aplurality of different molecular entities. Polypeptides may be preparedsynthetically in a peptide synthesizer and coupled to a carrier molecule(e.g., keyhole limpet hemocyanin) and injected over several months intorabbits. Rabbit sera is tested for immunoreactivity to the MTSpolypeptide or fragment. Monoclonal antibodies may be made by injectingmice with the protein polypeptides, fusion proteins or fragmentsthereof. Monoclonal antibodies will be screened by ELISA and tested forspecific immunoreactivity with MTS polypeptide or fragments thereof.See, Harlow & Lane, 1988. These antibodies will be useful in assays aswell as pharmaceuticals.

Once a sufficient quantity of desired polypeptide has been obtained, itmay be used for various purposes. A typical use is the production ofantibodies specific for binding. These antibodies may be eitherpolyclonal or monoclonal, and may be produced by in vitro or in vivotechniques well known in the art.

For production of polyclonal antibodies, an appropriate target immunesystem, typically mouse or rabbit, is selected. Substantially purifiedantigen is presented to the immune system in a fashion determined bymethods appropriate for the animal and by other parameters well known toimmunologists. Typical sites for injection are in footpads,intramuscularly, intraperitoneally, or intradermally. Of course, otherspecies may be substituted for mouse or rabbit. Polyclonal antibodiesare then purified using techniques known in the art, adjusted for thedesired specificity.

An immunological response is usually assayed with an immunoassay.Normally, such immunoassays involve some purification of a source ofantigen, for example, that produced by the same cells and in the samefashion as the antigen. A variety of immunoassay methods are well knownin the art. See, e.g., Harlow & Lane, 1988, or Goding, 1986.

Monoclonal antibodies with affinities of 10⁻⁸ M⁻¹ or preferably 10⁻⁹ to10⁻¹⁰ M⁻¹ or stronger will typically be made by standard procedures asdescribed, e.g., in Harlow & Lane, 1988 or Goding, 1986. Briefly,appropriate animals will be selected and the desired immunizationprotocol followed. After the appropriate period of time, the spleens ofsuch animals are excised and individual spleen cells fused, typically,to immortalized myeloma cells under appropriate selection conditions.Thereafter, the cells are clonally separated and the supernatants ofeach clone tested for their production of an appropriate antibodyspecific for the desired region of the antigen.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides, or alternatively, to selection of librariesof antibodies in phage or similar vectors. See Huse et al., 1989. Thepolypeptides and antibodies of the present invention may be used with orwithout modification. Frequently, polypeptides and antibodies will belabeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentagents, chemiluminescent agents, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant immunoglobulins may be produced (see U.S.Pat. No. 4,816,567).

“Binding partner” refers to a molecule capable of binding a ligandmolecule with high specificity, as for example, an antigen and anantigen-specific antibody or an enzyme and its inhibitor. In general,the specific binding partners must bind with sufficient affinity toimmobilize the analyte copy/complementary strand duplex (in the case ofpolynucleotide hybridization) under the isolation conditions. Specificbinding partners are known in the art and include, for example, biotinand avidin or streptavidin, IgG and protein A, the numerous, knownreceptor-ligand couples, and complementary polynucleotide strands. Inthe case of complementary polynucleotide binding partners, the partnersare normally at least about 15 bases in length, and may be at least 40bases in length. The polynucleotides may be composed of DNA, RNA, orsynthetic nucleotide analogs.

A “biological sample” refers to a sample of tissue or fluid suspected ofcontaining an analyte polynucleotide or polypeptide from an individualincluding, but not limited to, e.g., plasma, serum, spinal fluid, lymphfluid, the external sections of the skin, respiratory, intestinal, andgenito-urinary tracts, tears, saliva, blood cells, tumors, organs,tissue and samples of in vitro cell culture constituents.

As used herein, the terms “diagnosing” or “prognosing,” as used in thecontext of neoplasia, are used to indicate 1) the classification oflesions as neoplasia, 2) the determination of the severity of theneoplasia, or 3) the monitoring of the disease progression, prior to,during and after treatment.

“Encode”. A polynucleotide is said to “encode” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

“Isolated” or “substantially pure”. An “isolated” or “substantiallypure” nucleic acid (e.g., an RNA, DNA or a mixed polymer) is one whichis substantially separated from other cellular components whichnaturally accompany a native human sequence or protein, e.g., ribosomes,polymerases, many other human genome sequences and proteins. The termembraces a nucleic acid sequence or protein which has been removed fromits naturally occurring environment, and includes recombinant or clonedDNA isolates and chemically synthesized analogs or analogs biologicallysynthesized by heterologous systems.

“MTS Allele” refers to normal alleles of the MTS locus as well asalleles carrying variations that predispose individuals to developcancer of many sites including, for example, melanoma, ocular melanoma,leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin'slymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiocarcinoma,squamous cell carcinoma, CLL, and cancers of the pancreas, breast,brain, prostate, bladder, thyroid, ovary, uterus, testis, kidney,stomach, colon and rectum. Such predisposing alleles are also called“MTS susceptibility alleles”.

“MTS Locus,” “MTS gene,” “MTS Nucleic Acids”or “MTS Polynucleotide”refer to polynucleotides, all of which are in the MTS region, that arelikely to be expressed in normal tissue, certain alleles of whichpredispose an individual to develop melanoma and other cancers, such asocular melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma,Hodgkin's lymphoma, multiple myeloma, sarcoma, myosarcoma,cholangiocarcinoma, squamous cell carcinoma, CLL, and cancers of thepancreas, breast, brain, prostate, bladder, thyroid, ovary, uterus,testis, kidney, stomach, colon and rectum. The MTS locus is usedinterchangeably herein with the prior art designation MLM locus, and theuse of “MTS” is intended to include “MLM” as used with reference tolocus, gene, region, and the like. Mutations at the MTS locus may beinvolved in the initiation and/or progression of other types of tumors.The locus is indicated in part by mutations that predispose individualsto develop cancer. These mutations fall within the MTS region describedinfra. The MTS locus is intended to include coding sequences,intervening sequences and regulatory elements controlling transcriptionand/or translation. The MTS locus is intended to include all allelicvariations of the DNA sequence.

These terms, when applied to a nucleic acid, refer to a nucleic acidwhich encodes a MTS polypeptide (including p16), fragment, homolog orvariant, including, e.g., protein fusions or deletions. The nucleicacids of the present invention will possess a sequence which is eitherderived from, or substantially similar to a natural MTS-encoding gene orone having substantial homology with a natural MTS-encoding gene or aportion thereof. The coding sequence for an MTS polypeptide (MTS1) isshown in SEQ ID NO:1, and the amino acid sequence of an MTS polypeptide(MTS1) is shown in SEQ ID NO:2. The coding sequence for a second MTSpolypeptide (MTS1E1β) is shown in SEQ ID NO:13, and the correspondingamino acid sequence is shown in SEQ ID NO:14. The coding sequence for athird MTS polypeptide (MTS2) is shown in SEQ ID NO:15, and thecorresponding amino acid sequence is shown in SEQ ID NO:16. The term P16is used interchangeably with MTS1 and MTS1E1β and is used to mean bothMTS1 which encodes a p16 and MTS1E1β which encodes a p10. MTS1 andMTS1E1β are two forms of one gene, the two forms being dependent uponwhich of two promoters is used for transcription. MTS2 is a separateportion of the MTS region and it encodes a p15.

The polynucleotide compositions of this invention include RNA, cDNA,genomic DNA, synthetic forms, and mixed polymers, both sense andantisense strands, and may be chemically or biochemically modified ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those skilled in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

The present invention provides recombinant nucleic acids comprising allor part of the MTS region. The recombinant construct may be capable ofreplicating autonomously in a host cell. Alternatively, the recombinantconstruct may become integrated into the chromosomal DNA of the hostcell. Such a recombinant polynucleotide comprises a polynucleotide ofgenomic, cDNA, semi-synthetic, or synthetic origin which, by virtue ofits origin or manipulation, 1) is not associated with all or a portionof a polynucleotide with which it is associated in nature; 2) is linkedto a polynucleotide other than that to which it is linked in nature; or3) does not occur in nature.

Therefore, recombinant nucleic acids comprising sequences otherwise notnaturally occurring are provided by this invention. Although thewild-type sequence may be employed, it will often be altered, e.g., bydeletion, substitution or insertion.

cDNA or genomic libraries of various types may be screened as naturalsources of the nucleic acids of the present invention, or such nucleicacids may be provided by amplification of sequences resident in genomicDNA or other natural sources, e.g., by PCR. The choice of cDNA librariesnormally corresponds to a tissue source which is abundant in mRNA forthe desired proteins. Phage libraries are normally preferred, but othertypes of libraries may be used. Clones of a library are spread ontoplates, transferred to a substrate for screening, denatured and probedfor the presence of desired sequences.

The DNA sequences used in this invention will usually comprise at leastabout five codons (15 nucleotides), more usually at least about 7-15codons, and most preferably, at least about 35 codons. One or moreintrons may also be present. This number of nucleotides is usually aboutthe minimal length required for a successful probe that would hybridizespecifically with a MTS-encoding sequence.

Techniques for nucleic acid manipulation are described generally, forexample, in Sambrook et al., 1989 or Ausubel et al., 1992. Reagentsuseful in applying such techniques, such as restriction enzymes and thelike, are widely known in the art and commercially available from suchvendors as New England BioLabs, Boehringer Mannheim, Amersham, PromegaBiotec, U.S. Biochemicals, New England Nuclear, and a number of othersources. The recombinant nucleic acid sequences used to produce fusionproteins of the present invention may be derived from natural orsynthetic sequences. Many natural gene sequences are obtainable fromvarious cDNA or from genomic libraries using appropriate probes. See,GenBank, National Institutes of Health.

“MTS Region” refers to a portion of human chromosome 9 found in the P1clones P1-1062 and P1-1063. These P1 clones, in E. coli NS3529, weredeposited with the American Type Culture Collection, Rockville, Md.U.S.A. on Mar. 16, 1994 and assigned ATCC Nos. 69589 and 69590,respectively. This region contains the MTS locus, including the MTS1,MTS2 and MTS1E1β genes.

As used herein, the terms “MTS locus,” “MTS allele” and “MTS region” allrefer to the double-stranded DNA comprising the locus, allele, orregion, as well as either of the single-stranded DNAs comprising thelocus, allele or region.

As used herein, a “portion” of the MTS locus or region or allele isdefined as having a minimal size of at least about eight nucleotides, orpreferably about 15 nucleotides, or more preferably at least about 25nucleotides, and may have a minimal size of at least about 40nucleotides.

“MTS protein” or “MTS polypeptide” refer to a protein or polypeptideencoded by the MTS locus (including MTS1 polypeptide, MTS2 polypeptideand MTS1E1β polypeptide), variants or fragments thereof The term“polypeptide” refers to a polymer of amino acids and its equivalent anddoes not refer to a specific length of the product; thus, peptides,oligopeptides and proteins are included within the definition of apolypeptide. This term also does not refer to, or exclude modificationsof the polypeptide, for example, glycosylations, acetylations,phosphorylations, and the like. Included within the definition are, forexample, polypeptides containing one or more analogs of an amino acid(including, for example, unnatural amino acids, etc.), polypeptides withsubstituted linkages as well as other modifications known in the art,both naturally and non-naturally occurring. Ordinarily, suchpolypeptides will be at least about 50% homologous to the native MTSsequence, preferably in excess of about 90%, and more preferably atleast about 95% homologous. Also included are proteins encoded by DNAwhich hybridize under high or low stringency conditions, to MTS-encodingnucleic acids and closely related polypeptides or proteins retrieved byantisera to the MTS protein(s).

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acids, usually at least about 20 residues,more usually at least about 24 residues, typically at least about 28residues, and preferably more than about 35 residues.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. For instance, a promoter is operably linked to a codingsequence if the promoter affects its transcription or expression.

“Probes”. Polynucleotide polymorphisms associated with MTS alleles whichpredispose to certain cancers or are associated with most cancers aredetected by hybridization with a polynucleotide probe which forms astable hybrid with that of the target sequence, under stringent tomoderately stringent hybridization and wash conditions. If it isexpected that the probes will be perfectly complementary to the targetsequence, stringent conditions will be used. Hybridization stringencymay be lessened if some mismatching is expected, for example, ifvariants are expected with the result that the probe will not becompletely complementary. Conditions are chosen which rule outnonspecific/adventitious bindings, that is, which minimize noise. Sincesuch indications identify neutral DNA polymorphisms as well asmutations, these indications need further analysis to demonstratedetection of a MTS susceptibility allele.

Probes for MTS alleles may be derived from the sequences of the MTSregion or its cDNAs. The probes may be of any suitable length, whichspan all or a portion of the MTS region, and which allow specifichybridization to the MTS region. If the target sequence contains asequence identical to that of the probe, the probes may be short, e.g.,in the range of about 8-30 base pairs, since the hybrid will berelatively stable under even stringent conditions. If some degree ofmismatch is expected with the probe, i.e., if it is suspected that theprobe will hybridize to a variant region, a longer probe may be employedwhich hybridizes to the target sequence with the requisite specificity.

The probes will include an isolated polynucleotide attached to a labelor reporter molecule and may be used to isolate other polynucleotidesequences, having sequence similarity by standard methods. Fortechniques for preparing and labeling probes see, e.g., Sambrook et al.,1989 or Ausubel et al., 1992. Other similar polynucleotides may beselected by using homologous polynucleotides. Alternatively,polynucleotides encoding these or similar polypeptides may besynthesized or selected by use of the redundancy in the genetic code.Various codon substitutions may be introduced, e.g., by silent changes(thereby producing various restriction sites) or to optimize expressionfor a particular system. Mutations may be introduced to modify theproperties of the polypeptide, perhaps to change ligand-bindingaffinities, interchain affinities, or the polypeptide degradation orturnover rate.

Probes comprising synthetic oligonucleotides or other polynucleotides ofthe present invention may be derived from naturally occurring orrecombinant single- or double-stranded polynucleotides, or be chemicallysynthesized. Probes may also be labeled by nick translation, Klenowfill-in reaction, or other methods known in the art.

Portions of the polynucleotide sequence having at least about eightnucleotides, usually at least about 15 nucleotides, and fewer than about6 Kb, usually fewer than about 1.0 Kb, from a polynucleotide sequenceencoding MTS are preferred as probes. The probes may also be used todetermine whether mRNA encoding MTS is present in a cell or tissue.

“Protein modifications or fragments” are provided by the presentinvention for MTS polypeptides or fragments thereof which aresubstantially homologous to primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate unusual amino acids. Suchmodifications include, for example, acetylation, carboxylation,phosphorylation, glycosylation, ubiquitination, labeling, e.g., withradionuclides, and various enzymatic modifications, as will be readilyappreciated by those well skilled in the art. A variety of methods forlabeling polypeptides and of substituents or labels useful for suchpurposes are well known in the art, and include radioactive isotopessuch as ³²P, ligands, which bind to labeled antiligands (e.g.,antibodies), fluorophores, chemiluminescent agents, enzymes, andantiligands which can serve as specific binding pair members for alabeled ligand. The choice of label depends on the sensitivity required,ease of conjugation with the primer, stability requirements, andavailable instrumentation. Methods of labeling polypeptides are wellknown in the art. See, e.g., Sambrook et al., 1989 or Ausubel et al.,1992.

Besides substantially full-length polypeptides, the present inventionprovides for biologically active fragments of the polypeptides.Significant biological activities include ligand-binding, immunologicalactivity and other biological activities characteristic of MTSpolypeptides. Immunological activities include both immunogenic functionin a target immune system, as well as sharing of immunological epitopesfor binding, serving as either a competitor or substitute antigen for anepitope of the MTS protein. As used herein, “epitope” refers to anantigenic determinant of a polypeptide. An epitope could comprise threeamino acids in a spatial conformation which is unique to the epitope.Generally, an epitope consists of at least five such amino acids, andmore usually consists of at least 8-10 such amino acids. Methods ofdetermining the spatial conformation of such amino acids are known inthe art.

For immunological purposes, tandem-repeat polypeptide segments may beused as immunogens, thereby producing highly antigenic proteins.Alternatively, such polypeptides will serve as highly efficientcompetitors for specific binding. Production of antibodies specific forMTS polypeptides or fragments thereof is described below.

The present invention also provides for fusion polypeptides, comprisingMTS polypeptides and fragments. Homologous polypeptides may be fusionsbetween two or more MTS polypeptide sequences or between the sequencesof MTS and a related protein. Likewise, heterologous fusions may beconstructed which would exhibit a combination of properties oractivities of the derivative proteins. For example, ligand-binding orother domains may be “swapped” between different new fusion polypeptidesor fragments. Such homologous or heterologous fusion polypeptides maydisplay, for example, altered strength or specificity of binding. Fusionpartners include immunoglobulins, bacterial β-galactosidase, trpE,protein A, β-lactamase, alpha amylase, alcohol dehydrogenase and yeastalpha mating factor. See, e.g., Godowski et al., 1988.

Fusion proteins will typically be made by either recombinant nucleicacid methods, as described below, or may be chemically synthesized.Techniques for the synthesis of polypeptides are described, for example,in Merrifield, 1963.

“Protein purification” refers to various methods for the isolation ofthe MTS polypeptides from other biological material, such as from cellstransformed with recombinant nucleic acids encoding MTS, and are wellknown in the art. For example, such polypeptides may be purified byimmunoaffinity chromatography employing, e.g., the antibodies providedby the present invention. Various methods of protein purification arewell known in the art, and include those described in Deutscher, 1990and Scopes, 1982.

The terms “isolated”, “substantially pure”, and “substantiallyhomogeneous” are used interchangeably to describe a protein orpolypeptide which has been separated from components which accompany itin its natural state. A monomeric protein is substantially pure when atleast about 60 to 75% of a sample exhibits a single polypeptidesequence. A substantially pure protein will typically comprise about 60to 90% W/W of a protein sample, more usually about 95%, and preferablywill be over about 99% pure. Protein purity or homogeneity may beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis or a protein sample, followed byvisualizing a single polypeptide band upon staining the gel. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification utilized.

A MTS protein is substantially free of naturally associated componentswhen it is separated from the native contaminants which accompany it inits natural state. Thus, a polypeptide which is chemically synthesizedor synthesized in a cellular system different from the cell from whichit naturally originates will be substantially free from its naturallyassociated components. A protein may also be rendered substantially freeof naturally associated components by isolation, using proteinpurification techniques well known in the art.

A polypeptide produced as an expression product of an isolated andmanipulated genetic sequence is an “isolated polypeptide,” as usedherein, even if expressed in a homologous cell type. Synthetically madeforms or molecules expressed by heterologous cells are inherentlyisolated molecules.

“Recombinant nucleic acid” is a nucleic acid which is not naturallyoccurring, or which is made by the artificial combination of twootherwise separated segments of sequence. This artificial combination isoften accomplished by either chemical synthesis means, or by theartificial manipulation of isolated segments of nucleic acids, e.g., bygenetic engineering techniques. Such is usually done to replace a codonwith a redundant codon encoding the same or a conservative amino acid,while typically introducing or removing a sequence recognition site.Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a desired combination of functions.

“Regulatory sequences” refers to those sequences normally within 10 Kbof the coding region of a locus which affect the expression of the gene(including transcription of the gene, and translation, splicing,stability or the like of the messenger RNA).

“Substantial homology or similarity”. A nucleic acid or fragment thereofis “substantially homologous” (“or substantially similar”) to anotherif, when optimally aligned (with appropriate nucleotide insertions ordeletions) with the other nucleic acid (or its complementary strand),there is nucleotide sequence identity in at least about 60% of thenucleotide bases, usually at least about 70%, more usually at leastabout 80%, preferably at least about 90%, and more preferably at leastabout 95-98% of the nucleotide bases.

Alternatively, substantial homology or (similarity) exists when anucleic acid or fragment thereof will hybridize to another nucleic acid(or a complementary strand thereof) under selective hybridizationconditions, to a strand, or to its complement. Selectivity ofhybridization exists when hybridization which is substantially moreselective than total lack of specificity occurs. Typically, selectivehybridization will occur when there is at least about 55% homology overa stretch of at least about 14 nucleotides, preferably at least about65%, more preferably at least about 75%, and most preferably at leastabout 90%. See, Kanehisa, 1984. The length of homology comparison, asdescribed, may be over longer stretches, and in certain embodiments willoften be over a stretch of at least about nine nucleotides, usually atleast about 20 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about32 nucleotides, and preferably at least about 36 or more nucleotides.

Nucleic acid hybridization will be affected by such conditions as saltconcentration, temperature, or organic solvents, in addition to the basecomposition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess of30° C., typically in excess of 37° C., and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1000 mM,typically less than 500 mM, and preferably less than 200 mM. However,the combination of parameters is much more important than the measure ofany single parameter. See, e.g., Wetmur & Davidson, 1968.

Probe sequences may also hybridize specifically to duplex DNA undercertain conditions to form triplex or other higher order DNA complexes.The preparation of such probes and suitable hybridization conditions arewell known in the art.

The terms “substantial homology” or “substantial identity”, whenreferring to polypeptides, indicate that the polypeptide or protein inquestion exhibits at least about 30% identity with an entirenaturally-occurring protein or a portion thereof, usually at least about70% identity, and preferably at least about 95% identity.

“Substantially similar function” refers to the function of a modifiednucleic acid or a modified protein, with reference to the wild-type MTSnucleic acid or wild-type MTS polypeptide. The modified polypeptide willbe substantially homologous to the wild-type MTS polypeptide and willhave substantially the same function, i.e., the inhibition of Cdks,especially Cdk4. The modified polypeptide may have an altered amino acidsequence and/or may contain modified amino acids. In addition to thefunction of inhibiting Cdks, the modified polypeptide may have otheruseful properties, such as a longer half-life. The Cdk-inhibitoryactivity of the modified polypeptide may be substantially the same asthe activity of the wild-type MTS polypeptide. Alternatively, theCdk-inhibitory activity of the modified polypeptide may be higher thanhe activity of the wild-type MTS polypeptide. The modified polypeptideis synthesized using conventional techniques, or is encoded by amodified nucleic acid and produced using conventional techniques. Themodified nucleic acid is prepared by conventional techniques. A nucleicacid with a function substantially similar to the wild-type MTS genefunction produces the modified protein described above.

Homology, for polypeptides, is typically measured using sequenceanalysis software. See, e.g., the Sequence Analysis Software Package ofthe Genetics Computer Group, University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions, substitutions, and othermodifications. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine.

A polypeptide “fragment,” “portion” or “segment” is a stretch of aminoacid residues of at least about five to seven contiguous amino acids,often at least about seven to nine contiguous amino acids, typically atleast about nine to 13 contiguous amino acids and, most preferably, atleast about 20 to 30 or more contiguous amino acids.

The polypeptides of the present invention, if soluble, may be coupled toa solid-phase support, e.g., nitrocellulose, nylon, column packingmaterials (e.g., Sepharose beads), magnetic beads, glass wool, plastic,metal, polymer gels, cells, or other substrates. Such supports may takethe form, for example, of beads, wells, dipsticks, or membranes.

“Target region” refers to a region of the nucleic acid which isamplified and/or detected. The term “target sequence” refers to asequence with which a probe or primer will form a stable hybrid underdesired conditions.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, and immunology. See, e.g.,Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al., 1992;Glover, 1985; Anand, 1992; Guthrie & Fink, 1991. A general discussion oftechniques and materials for human gene mapping, including mapping ofhuman chromosome 9p, is provided, e.g., in White & Lalouel, 1988.

Preparation of Recombinant or Chemically Synthesized Nucleic Acids;Vectors, Transformation, Host Cells

Large amounts of the polynucleotides of the present invention may beproduced by replication in a suitable host cell. Natural or syntheticpolynucleotide fragments coding for a desired fragment will beincorporated into recombinant polynucleotide constructs, usually DNAconstructs, capable of introduction into and replication in aprokaryotic or eukaryotic cell. Usually the polynucleotide constructswill be suitable for replication in a unicellular host, such as yeast orbacteria, but may also be intended for introduction to (with and withoutintegration within the genome) cultured mammalian or plant or othereukaryotic cell lines. The purification of nucleic acids produced by themethods of the present invention are described, e.g., in Sambrook etal., 1989 or Ausubel et al., 1992.

The polynucleotides of the present invention may also be produced bychemical synthesis, e.g., by the phosphoramidite method described byBeaucage & Carruthers, 1981 or the triester method according toMatteucci et al., 1981, and may be performed on commercial, automatedoligonucleotide synthesizers. A double-stranded fragment may be obtainedfrom the single-stranded product of chemical synthesis either bysynthesizing the complementary strand and annealing the strands togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

Polynucleotide constructs prepared for introduction into a prokaryoticor eukaryotic host may comprise a replication system recognized by thehost, including the intended polynucleotide fragment encoding thedesired polypeptide, and will preferably also include transcription andtranslational initiation regulatory sequences operably linked to thepolypeptide encoding segment. Expression vectors may include, forexample, an origin of replication or autonomously replicating sequence(ARS) and expression control sequences, a promoter, an enhancer andnecessary processing information sites, such as ribosome-binding sites,RNA splice sites, polyadenylation sites, transcriptional terminatorsequences, and mRNA stabilizing sequences. Secretion signals may also beincluded where appropriate, whether from a native MTS protein or fromother receptors or from secreted polypeptides of the same or relatedspecies, which allow the protein to cross and/or lodge in cellmembranes, and thus attain its functional topology, or be secreted fromthe cell. Such vectors may be prepared by means of standard recombinanttechniques well known in the art and discussed, for example, in Sambrooket al., 1989 or Ausubel et al. 1992.

The selection of an appropriate promoter and other necessary vectorsequences will be selected so as to be functional in the host, and mayinclude, when appropriate, those naturally associated with MTS genes.Examples of workable combinations of cell lines and expression vectorsare described in Sambrook et al., 1989 or Ausubel et al., 1992; seealso, e.g., Metzger et al., 1988. Many useful vectors are known in theart and may be obtained from such vendors as Stratagene, New EnglandBiolabs, Promega Biotech, and others. Promoters such as the trp, lac andphage promoters, tRNA promoters and glycolytic enzyme promoters may beused in prokaryotic hosts. Useful yeast promoters include promoterregions for metallothionein, 3-phosphoglycerate kinase or otherglycolytic enzymes such as enolase or glyceraldehyde-3-phosphatedehydrogenase, enzymes responsible for maltose and galactoseutilization, and others. Vectors and promoters suitable for use in yeastexpression are further described in Hitzeman et al., EP 73,675A.Appropriate non-native mammalian promoters might include the early andlate promoters from SV40 (Fiers et al., 1978) or promoters derived frommurine molony leukemia virus, mouse tumor virus, avian sarcoma viruses,adenovirus II, bovine papilloma virus or polyoma. In addition, theconstruct may be joined to an amplifiable gene (e.g., DHFR) so thatmultiple copies of the gene may be made. For appropriate enhancer andother expression control sequences, see also Enhancers and EukaryoticGene Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1983).

While such expression vectors may replicate autonomously, they may alsoreplicate by being inserted into the genome of the host cell, by methodswell known in the art.

Expression and cloning vectors will likely contain a selectable marker,a gene encoding a protein necessary for survival or growth of a hostcell transformed with the vector. The presence of this gene ensuresgrowth of only those host cells which express the inserts. Typicalselection genes encode proteins that a) confer resistance to antibioticsor other toxic substances, e.g. ampicillin, neomycin, methotrexate,etc., b) complement auxotrophic deficiencies, or c) supply criticalnutrients not available from complex media, e.g., the gene encodingD-alanine racemase for Bacilli. The choice of the proper selectablemarker will depend on the host cell, and appropriate markers fordifferent hosts are well known in the art.

The vectors containing the nucleic acids of interest can be transcribedin vitro, and the resulting RNA introduced into the host cell bywell-known methods, e.g., by injection (see, T. Kubo et al., 1988), orthe vectors can be introduced directly into host cells by methods wellknown in the art, which vary depending on the type of cellular host,including electroporation; transfection employing calcium chloride,rubidium chloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; infection (where the vector isan infectious agent, such as a retroviral genome); and other methods.See generally, Sambrook et al., 1989 and Ausubel et al., 1992. Theintroduction of the polynucleotides into the host cell by any methodknown in the art, including, inter alia, those described above, will bereferred to herein as “transformation.” The cells into which have beenintroduced nucleic acids described above are meant to also include theprogeny of such cells.

Large quantities of the nucleic acids and polypeptides of the presentinvention may be prepared by expressing the MTS nucleic acids orportions thereof in vectors or other expression vehicles in compatibleprokaryotic or eukaryotic host cells. The most commonly used prokaryotichosts are strains of Escherichia coli, although other prokaryotes, suchas Bacillus subtilis or Pseudomonas may also be used.

Mammalian or other eukaryotic host cells, such as those of yeast,filamentous fungi, plant, insect, or amphibian or avian species, mayalso be useful for production of the proteins of the present invention.Propagation of mammalian cells in culture is per se well known. See,Jakoby and Pastan (eds.), 1979. Examples of commonly used mammalian hostcell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells,and WI38, BHK, and COS cell lines, although it will be appreciated bythe skilled practitioner that other cell lines may be appropriate, e.g.,to provide higher expression, desirable glycosylation patterns, or otherfeatures.

Clones are selected by using markers depending on the mode of the vectorconstruction. The marker may be on the same or a different DNA molecule,preferably the same DNA molecule. In prokaryotic hosts, the transformantmay be selected, e.g., by resistance to ampicillin, tetracycline orother antibiotics. Production of a particular product based ontemperature sensitivity may also serve as an appropriate marker.

Prokaryotic or eukaryotic cells transformed with the polynucleotides ofthe present invention will be useful not only for the production of thenucleic acids and polypeptides of the present invention, but also, forexample, in studying the characteristics of MTS polypeptides.

Antisense polynucleotide sequences are useful in preventing ordiminishing the expression of the MTS locus, as will be appreciated bythose skilled in the art. For example, polynucleotide vectors containingall or a portion of the MTS locus or other sequences from the MTS region(particularly those flanking the MTS locus) may be placed under thecontrol of a promoter in an antisense orientation and introduced into acell. Expression of such an antisense construct within a cell willinterfere with MTS transcription and/or translation and/or replication.

Cycline and Cdks are ubiquitous cell-cycle control elements ineukaryotes. Such proteins were initially discovered in yeast, and havebeen found in marine invertebrates, amphibians and mammals, includingmouse, rabbit and humans. Homologous cell-cycle control genes areidentified in other species by using probes and/or primers based on thegene sequence in one species. Thus, probes and primers based on the MTSgene sequences disclosed herein are used to identify homologous MTS genesequences and proteins in other species. These MTS gene sequences andproteins are used in the diagnostic/prognostic, therapeutic and drugscreening methods described herein for the species from which they havebeen isolated.

Methods of Use: Nucleic Acid Diagnosis and Diagnostic Kits

In order to detect the presence of a MTS allele predisposing anindividual to cancer, a biological sample such as blood is prepared andanalyzed for the presence or absence of susceptibility alleles of MTS.In order to detect the presence of neoplasia, the progression towardmalignancy of a precursor lesion, or as a prognostic indicator, abiological sample of the lesion is prepared and analyzed for thepresence or absence of neoplastic alleles of MTS. Results of these testsand interpretive information are returned to the health care providerfor communication to the tested individual. Such diagnoses may beperformed by diagnostic laboratories, or, alternatively, diagnostic kitsare manufactured and sold to health care providers or to privateindividuals for self-diagnosis.

Initially, the screening method involves amplification of the relevantMTS sequences, e.g., by PCR, followed by DNA sequence analysis. Inanother preferred embodiment of the invention, the screening methodinvolves a non-PCR based strategy. Such screening methods includetwo-step label amplification methodologies that are well known in theart. Both PCR and non-PCR based screening strategies can detect targetsequences with a high level of sensitivity.

The most popular method used today is target amplification. Here, thetarget nucleic acid sequence is amplified with polymerases. Oneparticularly preferred method using polymerase-driven amplification isthe polymerase chain reaction (PCR). A preferred PCR based strategycontemplated within the scope of this invention is provided inExample 1. The polymerase chain reaction and other polymerase-drivenamplification assays can achieve over a million-fold increase in copynumber through the use of polymerase-driven amplification cycles. Onceamplified, the resulting nucleic acid can be sequenced or used as asubstrate for DNA probes.

When the probes are used to detect the presence of the target sequences(for example, in screening for cancer susceptibility), the biologicalsample to be analyzed, such as blood or serum, may be treated, ifdesired, to extract the nucleic acids. The sample nucleic acid may beprepared in various ways to facilitate detection of the target sequence;e.g. denaturation, restriction digestion, electrophoresis or dotblotting. The targeted region of the analyte nucleic acid usually mustbe at least partially single-stranded to form hybrids with the targetingsequence of the probe. If the sequence is naturally single-stranded,denaturation will not be required. However, if the sequence isdouble-stranded, the sequence will probably need to be denatured.Denaturation can be carried out by various techniques known in the art.

Analyte nucleic acid and probe are incubated under conditions whichpromote stable hybrid formation of the target sequence in the probe withthe putative targeted sequence in the analyte. The region of the probeswhich is used to bind to the analyte can be made completelycomplementary to the targeted region of human chromosome 9p. Therefore,high stringency conditions are desirable in order to prevent falsepositives. However, conditions of high stringency are used only if theprobes are complementary to regions of the chromosome which are uniquein the genome. The stringency of hybridization is determined by a numberof factors during hybridization and during the washing procedure,including temperature, ionic strength, base composition, probe length,and concentration of formamide. These factors are outlined in, forexample, Maniatis et al., 1982 and Sambrook et al., 1989. Under certaincircumstances, the formation of higher order hybrids, such as triplexes,quadraplexes, etc., may be desired to provide the means of detectingtarget sequences.

Detection, if any, of the resulting hybrid is usually accomplished bythe use of labeled probes. Alternatively, the probe may be unlabeled,but may be detectable by specific binding with a ligand which islabeled, either directly or indirectly. Suitable labels, and methods forlabeling probes and ligands are known in the art, and include, forexample, radioactive labels which may be incorporated by known methods(e.g., nick translation, random priming or kinasing), biotin,fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies and the like.Variations of this basic scheme are known in the art, and include thosevariations that facilitate separation of the hybrids to be detected fromextraneous materials and/or that amplify the signal from the labeledmoiety. A number of these variations are reviewed in, e.g., Matthews &Kricka, 1988; Landegren et al., 1988; Mittlin, 1989; U.S. Pat. No.4,868,105, and in EPO Publication No. 225,807.

As noted above, non-PCR based screening assays are also contemplated inthis invention. An exemplary non-PCR based procedure is provided inExample 15. This procedure hybridizes a nucleic acid probe (or an analogsuch as a methyl phosphonate backbone replacing the normalphosphodiester), to the low level DNA target. This probe may have anenzyme covalently linked to the probe, such that the covalent linkagedoes not interfere with the specificity of the hybridization. Thisenzyme-probe-conjugate-target nucleic acid complex can then be isolatedaway from the free probe enzyme conjugate and a substrate is added forenzyme detection. Enzymatic activity is observed as a change in colordevelopment or luminescent output resulting in a 10³-10⁶ increase insensitivity. For an example relating to the preparation ofoligodeoxynucleotide-alkaline phosphatase conjugates and their use ashybridization probes see Jablonski et al., 1986.

Two-step label amplification methodologies are known in the art. Theseassays work on the principle that a small ligand (such as digoxigenin,biotin, or the like) is attached to a nucleic acid probe capable ofspecifically binding MTS. An exemplary probe for MTS1 is the nucleicacid probe corresponding to nucleotide positions 448 to 498 of SEQ IDNO:4. Allele specific probes are also contemplated within the scope ofthis example and exemplary allele specific probes include probesencompassing the predisposing mutations summarized in Table 3 and thesomatic mutations in tumors summarized in Table 5.

In one example, the small ligand attached to the nucleic acid probe isspecifically recognized by an antibody-enzyme conjugate. In oneembodiment of this example, digoxigenin is attached to the nucleic acidprobe. Hybridization is detected by an antibody-alkaline phosphataseconjugate which turns over a chemiluminescent substrate. For methods forlabeling nucleic acid probes according to this embodiment see Martin etal., 1990. In a second example, the small ligand is recognized by asecond ligand-enzyme conjugate that is capable of specificallycomplexing to the first ligand. A well known embodiment of this exampleis the biotin-avidin type of interactions. For methods for labelingnucleic acid probes and their use in biotin-avidin based assays seeRigby, et al., 1977 and Nguyen, et al. (1992).

It is also contemplated within the scope of this invention that thenucleic acid probe assays of this invention will employ a cocktail ofnucleic acid probes capable of detecting MTS genes. Thus, in one exampleto detect the presence of MTS1 in a cell sample, more than one probecomplementary to MTS1 is employed and in particular the number ofdifferent probes is alternatively 2, 3, or 5 different nucleic acidprobe sequences. In another example, to detect the presence of mutationsin the MTS1 gene sequence in a patient, more than one probecomplementary to MTS1 is employed where the cocktail includes probescapable of binding to the allele-specific mutations identified inpopulations of patients with alterations in MTS1. In this embodiment,any number of probes can be used, and will preferably include probescorresponding to the major gene mutations identified as predisposing anindividual to breast cancer. Some candidate probes contemplated withinthe scope of the invention include probes that include theallele-specific mutations identified in Tables 3 and 5.

Methods of Use: Peptide Diagnosis and Diagnostic Kits

The neoplastic condition of lesions can also be detected on the basis ofthe alteration of wild-type MTS polypeptide. Such alterations can bedetermined by sequence analysis in accordance with conventionaltechniques. More preferably, antibodies (polyclonal or monoclonal) areused to detect differences in, or the absence of MTS peptides. In apreferred embodiment of the invention, antibodies will immunoprecipitateMTS proteins from solution as well as react with MTS protein on Westernor immunoblots of polyacrylamide gels. In another preferred embodiment,antibodies will detect MTS proteins in paraffin or frozen tissuesections, using immunocytochemical techniques. Techniques for raisingand purifying antibodies are well known in the art, and any suchtechniques may be chosen to achieve the preparation of the invention.

Preferred embodiments relating to methods for detecting MTS or itsmutations include enzyme linked immunosorbent assays (ELISA),radioimmunoassays (RIA), immunoradiometric assays (IRMA) andimmunoenzymatic assays (IEMA), including sandwich assays usingmonoclonal and/or polyclonal antibodies. Exemplary sandwich assays aredescribed by David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530,hereby incorporated by reference, and exemplified in Example 18.

Methods of Use: Drug Screening

The present invention is particularly useful for screening compounds byusing the Cdk polypeptides or binding fragments thereof in any of avariety of drug screening techniques. Preferably, Cdk4 is utilized. TheCdk polypeptide or fragment employed in such a test may either be freein solution, affixed to a solid support, or borne on a cell surface. Onemethod of drug screening utilizes eukaryotic or prokaryotic host cellswhich are stably transformed with recombinant polynucleotides expressingthe polypeptide or fragment, preferably in competitive binding assays.Such cells, either in viable or fixed form, can be used for standardbinding assays. One may measure, for example, for the formation ofcomplexes between a Cdk polypeptide or fragment and the agent beingtested, or examine the degree to which the formation of a complexbetween a Cdk polypeptide or fragment and MTS polypeptide or fragment isinterfered with by the agent being tested.

Thus, the present invention provides methods of screening for drugscomprising contacting such an agent with a Cdk polypeptide or fragmentthereof and assaying: 1) for the presence of a complex between the agentand the Cdk polypeptide or fragment, or 2) for the presence of a complexbetween the Cdk polypeptide or fragment and a ligand, by methods wellknown in the art. The activity of Cdk is also measured to determine ifthe agent is capable of inhibiting Cdk, and hence capable of regulatingthe cell cycle. In such competitive binding assays the Cdk polypeptideor fragment is typically labeled. Free Cdk polypeptide or fragment isseparated from that present in a protein:protein complex, and the amountof free (i.e., uncomplexed) label is a measure of the binding of theagent being tested to Cdk or its interference with Cdk:MTS polypeptidebinding, respectively. Small peptides of MTS polypeptide (peptidemimetics) are analyzed in this manner to identify those which have Cdkinhibitory activity.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the Cdk polypeptidesand is described in detail in Geysen, published application WO 84/03564,published Sep. 13, 1984. Briefly stated, large numbers of differentsmall peptide test compounds are synthesized on a solid substrate, suchas plastic pins or some other surface. The peptide test compounds arereacted with Cdk polypeptide and washed. Bound Cdk polypeptide is thendetected by methods well known in the art.

Purified Cdk can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to the polypeptide can be used to capture antibodies toimmobilize the Cdk polypeptide on the solid phase.

The present invention also contemplates the use of competitive drugscreening assays, in which neutralizing antibodies capable ofspecifically binding the Cdk polypeptide compete with a test compoundfor binding to the Cdk polypeptide or fragments thereof. In this manner,the antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants of the Cdk polypeptide.

A further technique for drug screening involves the use of hosteukaryotic cell lines or cells (such as described above) which have anonfunctional MTS gene. These host cell lines or cells are defective incell cycle control at the Cdk level. The host cell lines or cells aregrown in the presence of drug compound. The rate of growth of the hostcells is measured to determine if the compound is capable of regulatingthe cell cycle. One means of measuring the growth rate is by determiningthe biological activity of the Cdks, preferably Cdk4.

Methods of Use: Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact (e.g., agonists, antagonists, inhibitors) in orderto fashion drugs which are, for example, more active or stable forms ofthe polypeptide, or which, e.g., enhance or interfere with the functionof a polypeptide in vivo. See, e.g., Hodgson, 1991. In one approach, onefirst determines the three-dimensional structure of a protein ofinterest (e.g., p16 or Cdk4) or, for example, of the Cdk4-p16 complex,by x-ray crystallography, by computer modeling or most typically, by acombination of approaches. Less often, useful information regarding thestructure of a polypeptide may be gained by modeling based on thestructure of homologous proteins. An example of rational drug design isthe development of HIV protease inhibitors (Erickson et al., 1990). Inaddition, peptides (e.g., p16 or Cdk4) are analyzed by an alanine scan(Wells, 1991). In this technique, an amino acid residue is replaced byAla, and its effect on the peptide's activity is determined. Each of theamino acid residues of the peptide is analyzed in this manner todetermine the important regions of the peptide.

It is also possible to isolate a target-specific antibody, selected by afunctional assay, and then to solve its crystal structure. In principle,this approach yields a pharmacore upon which subsequent drug design canbe based. It is possible to bypass protein crystallography altogether bygenerating anti-idiotypic antibodies (anti-ids) to a functional,pharmacologically active antibody. As a mirror image of a mirror image,the binding site of the anti-ids would be expected to be an analog ofthe original receptor. The anti-id could then be used to identify andisolate peptides from banks of chemically or biologically produced banksof peptides. Selected peptides would then act as the pharmacore.

Thus, one may design drugs which have, e.g., improved MTS activity orstability or which act as inhibitors, agonists, antagonists, etc. of MTSactivity. By virtue of the availability of cloned MTS sequences,sufficient amounts of the MTS polypeptide may be made available toperform such analytical studies as x-ray crystallography. In addition,the knowledge of the MTS protein sequence provided herein will guidethose employing computer modeling techniques in place of, or in additionto x-ray crystallography.

Methods of Use: Gene Therapy

According to the present invention, a method is also provided ofsupplying wild-type MTS function to a cell which carries mutant MTSalleles. Supplying such a function should suppress neoplastic growth ofthe recipient cells. The wild-type MTS gene or a part of the gene may beintroduced into the cell in a vector such that the gene remainsextrachromosomal. In such a situation, the gene will be expressed by thecell from the extrachromosomal location. If a gene portion is introducedand expressed in a cell carrying a mutant MTS allele, the gene portionshould encode a part of the MTS protein which is required fornon-neoplastic growth of the cell. More preferred is the situation wherethe wild-type MTS gene or a part thereof is introduced into the mutantcell in such a way that it recombines with the endogenous mutant MTSgene present in the cell. Such recombination requires a doublerecombination event which results in the correction of the MTS genemutation. Vectors for introduction of genes both for recombination andfor extrachromosomal maintenance are known in the art, and any suitablevector may be used. Methods for introducing DNA into cells such aselectroporation, calcium phosphate coprecipitation and viraltransduction are known in the art, and the choice of method is withinthe competence of the routineer. Cells transformed with the wild-typeMTS gene can be used as model systems to study cancer remission and drugtreatments which promote such remission.

As generally discussed above, the MTS gene or fragment, whereapplicable, may be employed in gene therapy methods in order to increasethe amount of the expression products of such genes in cancer cells.Such gene therapy is particularly appropriate for use in both cancerousand pre-cancerous cells, in which the level of MTS polypeptide is absentor diminished compared to normal cells. It may also be useful toincrease the level of expression of a given MTS gene even in those tumorcells in which the mutant gene is expressed at a “normal” level, but thegene product is not fully functional.

Gene therapy would be carried out according to generally acceptedmethods, for example, as described by Friedman in Therapy for GeneticDisease, T. Friedman, ed., Oxford University Press (1991), pp. 105-121.Cells from a patient's tumor would be first analyzed by the diagnosticmethods described above, to ascertain the production of MTS polypeptidein the tumor cells. A 30 virus or plasmid vector, containing a copy ofthe MTS gene linked to expression control elements and capable ofreplicating inside the tumor cells, is prepared. Suitable vectors areknown, such as disclosed in U.S. Pat. No. 5,252,479 and PCT publishedapplication WO 93/07282. The vector is then injected into the patient,either locally at the site of the tumor or systemically (in order toreach any tumor cells that may have metastasized to other sites). If thetransfected gene is not permanently incorporated into the genome of eachof the targeted tumor cells, the treatment may have to be repeatedperiodically. Since MTS polypeptides are intimately involved in thecontrol of the cell cycle, it is preferred that the MTS gene beintroduced with its own regulatory elements, to avoid constitutiveexpression of MTS polypeptide by all cells which take up the gene.

Gene transfer systems known in the art may be useful in the practice ofthe gene therapy methods of the present invention. These include viraland nonviral transfer methods. A number of viruses have been used asgene transfer vectors, including papovaviruses (e.g., SV40, Madzak etal., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorzigliaand Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992;Wilkinson et al., 1992; Stratford-Perricaudet et al., 1990), vacciniavirus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohi et al.,1990), herpesviruses including HSV and EBV (Margolskee, 1992; Johnson etal., 1992; Fink et al., 1992; Breakfield and Geller, 1987; Freese etal., 1990), and retroviruses of avian (Brandyopadhyay and Temin, 1984;Petropoulos et al., 1992), murine (Miller, 1992; Miller et al., 1985;Sorge et al., 1984; Mann and Baltimore, 1985; Miller et al., 1988), andhuman origin (Shimada et al., 1991; Helseth et al., 1990; Page et al.,1990; Buchschacher and Panganiban, 1992). Most human gene therapyprotocols have been based on disabled murine retroviruses.

Nonviral gene transfer methods known in the art include chemicaltechniques such as calcium phosphate coprecipitation (Graham and van derEb, 1973; Pellicer et al., 1980); mechanical techniques, for examplemicroinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster etal., 1981; Constantini and Lacy, 1981); membrane fusion-mediatedtransfer via liposomes (Felgner et al., 1987; Wang and Huang, 1989;Kaneda et al, 1989; Stewart et al., 1992; Nabel et al., 1990; Lim etal., 1992); and direct DNA uptake and receptor-mediated DNA transfer(Wolff et al., 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al.,1989b; Wolff et al., 1991; Wagner et al., 1990; Wagner et al., 1991;Cotten et al., 1990; Curiel et al., 1991a; Curiel et al., 1991b).Viral-mediated gene transfer can be combined with direct in vivo genetransfer using liposome delivery, allowing one to direct the viralvectors to the tumor cells and not into the surrounding nondividingcells. Alternatively, the retroviral vector producer cell line can beinjected into tumors (Culver et al., 1992). Injection of producer cellswould then provide a continuous source of vector particles. Thistechnique has been approved for use in humans with inoperable braintumors.

In an approach which combines biological and physical gene transfermethods, plasmid DNA of any size is combined with apolylysine-conjugated antibody specific to the adenovirus hexon protein,and the resulting complex is bound to an adenovirus vector. Thetrimolecular complex is then used to infect cells. The adenovirus vectorpermits efficient binding, internalization, and degradation of theendosome before the coupled DNA is damaged.

Liposome/DNA complexes have been shown to be capable of mediating directin vivo gene transfer. While in standard liposome preparations the genetransfer process is nonspecific, localized in vivo uptake and expressionhave been reported in tumor deposits, for example, following direct insitu administration (Nabel, 1992).

Methods of Use: Peptide Therapy

Peptides which have MTS activity can be supplied to cells which carrymutant or missing MTS alleles. The sequences of the MTS proteins aredisclosed (SEQ ID NO:2, SEQ ID NO:14 and SEQ ID NO:16). Protein can beproduced by expression of the cDNA sequence in bacteria, for example,using known expression vectors. Alternatively, MTS polypeptide can beextracted from MTS-producing mammalian cells. In addition, thetechniques of synthetic chemistry can be employed to synthesize MTSprotein. Any of such techniques can provide the preparation of thepresent invention which comprises the MTS protein. The preparation issubstantially free of other human proteins. This is most readilyaccomplished by synthesis in a microorganism or in vitro.

Active MTS molecules can be introduced into cells by microinjection orby use of liposomes, for example. Alternatively, some active moleculesmay be taken up by cells, actively or by diffusion. Extracellularapplication of the MTS gene product may be sufficient to affect tumorgrowth. Supply of molecules with MTS activity should lead to partialreversal of the neoplastic state. Other molecules with MTS activity (forexample, peptides, drugs or organic compounds) may also be used toeffect such a reversal. Modified polypeptides having substantiallysimilar function are also used for peptide therapy.

Methods of Use: Transformed Hosts

Similarly, cells and animals which carry a mutant MTS allele can be usedas model systems to study and test for substances which have potentialas therapeutic agents. The cells are typically cultured epithelialcells. These may be isolated from individuals with MTS mutations, eithersomatic or germline. Alternatively, the cell line can be engineered tocarry the mutation in the MTS allele, as described above. After a testsubstance is applied to the cells, the neoplastically transformedphenotype of the cell is determined. Any trait of neoplasticallytransformed cells can be assessed, including anchorage-independentgrowth, tumorigenicity in nude mice, invasiveness of cells, and growthfactor dependence. Assays for each of these traits are known in the art.

Animals for testing therapeutic agents can be selected after mutagenesisof whole animals or after treatment of germline cells or zygotes. Suchtreatments include insertion of mutant MTS alleles, usually from asecond animal species, as well as insertion of disrupted homologousgenes. Alternatively, the endogenous MTS gene(s) of the animals may bedisrupted by insertion or deletion mutation or other genetic alterationsusing conventional techniques (Capecchi, 1989; Valancius and Smithies,1991; Hasty et al., 1991; Shinkai et al., 1992; Mombaerts et al., 1992;Philpott et al., 1992; Snouwaert et al., 1992; Donehower et al., 1992).After test substances have been administered to the animals, the growthof tumors must be assessed. If the test substance prevents or suppressesthe growth of tumors, then the test substance is a candidate therapeuticagent for the treatment of the cancers identified herein.

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

EXAMPLE 1 Materials and Methods

A. MTS Pedigrees

FIGS. 1A-1D show Kindreds 3137, 3161, 3355 and 1771, respectively. Theoccurrence of cancer in these kindreds is shown in the Figures. Allmelanomas in kindred 3137 carry the susceptible haplotype, and othercancers carrying the susceptible haplotype are also shown for thiskindred. All melanomas in kindreds 3161 and 3355 carry the susceptiblehaplotype. A mutation in MTS was identified for the cancers in kindred1771.

B. Tumor Cell Lines

Seventy-six melanoma cell lines were obtained from the Ludwig Institutefor Cancer Research, Memorial Sloan-Kettering Cancer Center, and 8melanoma cell lines and five non-melanoma lines from the American TypeCulture Collection (ATCC).

C. Preparation and Analysis of Tumor Cell Line DNA

DNA was isolated from cell lines by the addition of approximately 1×10⁷cells to 3 ml lysis buffer (0.1 M NaCl; 0.1M TrisHCl pH8.0; 5 mM EDTA;0.5% SDS), followed by vortexing and incubation at 65° C. for 30minutes. 0.5 ml of 8M KOAc was added, and the reaction was mixed andincubated on ice for 30 minutes. After centrifugation (five minutes at10,000×g), the supernatant was precipitated with an equal volume of 95%ethanol and centrifuged again (15 minutes at 10,000×g). The DNA wasresuspended in 50-200 ml H₂O.

D. PCR Reactions

50 ng template was added to 30 pmol of each oligonucleotide primer in a20 ml reaction mixture that contained 0.1 mM dNTPs, 10 mM Tris-HCl(pH8.3), 50 mM KCl, 2 mM MgCl₂, 0.01% gelatin, and 1 unit Amplitaqpolymerase (Perkin-Elmer). Samples were cycled in a Perkin-Elmer 9600thermal cycler 35 times at 94° C. for 10 seconds, 55° C. for 10 seconds,and 72° C. for 10 seconds. The products were visualized afterelectrophoresis through either 1.5% agarose (SeaKem) or 3% NuSieve 3:1agarose (FMC BioProducts) by ethidium bromide staining.

E. YACs

Yeast artificial chromosomes (YACs) containing markers in the MTS regionwere obtained by screening the CEPH YAC libraries with IFNA, D9S171, andD9S126 using PCR conditions described above. Yeast strains containingYACs were grown at 30° C. for three days with vigorous shaking in AHCmedium (10 g/l casein hydrolysate-acid; 1.7 g/l yeast nitrogen base; 5g/l ammonium sulfate; 20 mg/l adenine hemisulfate; 2% glucose; pH=5.8).Yeast DNA was prepared as described by Ausubel et al., 1992.

F. Phage Library Construction

Yeast genomic DNA containing YAC DNA was digested to completion withBamHI, inserted into BamHI-digested EMBL3 phage arms (Promega) using T4DNA ligase (Boehringer-Mannheim), and packaged in vitro with Gigapack IIextracts (Stratagene). Phage were grown on E coli strain C600.Recombinant phage containing human DNA were identified by hybridizationwith ³²P-labeled human C_(o)t-1 DNA (GIBCO-BRL). Phage including humansequences joined to YAC vector (end clones) were identified by screeningwith PCR fragments containing sequences from the YAC left or right arm.Hybridization and washing were carried out under standard conditions(Middleton et al., 1991). Positive plaques were picked and purified byreplating three times. Phage DNA was prepared using Qiaex columns(Qiagen).

G. Cosmid Library Construction

Yeast genomic DNA containing YAC DNA was digested partially with Sau3Aand fractionated by size on a linear (10-40%) sucrose gradient, asdescribed in Maniatis et al., 1982. SuperCos 1 cosmid vector(Stratagene) was prepared according to manufacturer's directions, mixedwith insert DNA at a mass ratio of 4:1 (insert:vector), treated withligase, and packaged in vitro, as described above. Cosmids wereintroduced into DH5α host cells and plated at a density of 2000 coloniesper 15 cm petri dish. Colony hybridization was carried out as describedabove and in Maniatis et al., 1982.

H. P1 Clones

P1 clones spanning the MTS region were obtained from Genome Systems,Inc., St. Louis, Mo., by screening with STSs prepared as describedherein. DNA from these clones was isolated by alkaline lysis (Birnboimand Doly, 1979), followed by cesium chloride gradient centrifugation(Maniatis et al., 1982).

I. Generation of STSs

STSs were generated by sequencing 1.0 mg of P1, cosmid, or template DNAwith oligonucleotides complementary to sequences flanking the cloningsite of the P1 vector (pSacBII), SuperCos 1 vector, or the EMBL3 vector.Sequencing was done on an ABI 373A DNA sequencing system with the PRISMReady Reaction DyeDeoxy Terminator Cycle Sequencing Kit (ABI). STSs weredesigned to be as close as possible to 20 bp long and to have a Tm asclose as possible to 60° C.

J. Germline Mutations in MTS1 in Melanoma-Prone Kindreds

Genomic DNA from carrier individuals was prepared from blood usingstandard methods. Primers were designed at intron positions to amplifycoding exons 1 or 2 from MTS1, or coding exon 2 from MTS2 using 20 ngDNA from each sample. PCR reactions used the standard buffer, exceptDMSO was added to a final concentration of 5%. Cycle sequencingreactions were carried out using α-P³²-dATP on the amplified productsusing primers positioned at different points in the sequence. Thesequencing products were analyzed on 6% denaturing polyacrylamide gelsby loading all the (A) reactions side by side, followed by the (C)reactions, etc. All polymorphisms were confirmed by sequence analysis ofthe opposite strand.

EXAMPLE 2 Localization of MTS Using Genetically Linked Markers

To analyze tumor cell lines for homozygous deletions in the 9p21 region,a set of markers known to be linked to MTS was utilized. These markerswere used originally to demonstrate dramatic linkage (LOD score=12.7) ofmelanoma predisposition in 10 Utah kindreds and one Texas kindred(Cannon-Albright et al., 1992). The markers included a sequence from theα-interferon gene cluster (IFNA) (Kwiatkowski & Diaz, 1992) which wasthe most distal marker tested, a proximal marker (D9S104), and fouradditional markers in between (D9S171, D9S126, D9S161, and D9S169)(Cannon-Albright et al., 1992). From genetic studies, the linearsequence of the intervening markers was thought to be: D9S171, D9S126,D9S161, D9S169. The IFNA marker consisted of an oligonucleotide primerpair that amplified two fragments from wildtype genomic DNA: a roughly138-150 bp polymorphic fragment (IFNA-1) that contains a poly(CA)stretch, and a roughly 120 bp invariant fragment (IFNA-s). The locationof IFNA-s with respect to IFNA-1 was unknown.

Five non-melanoma tumor cell lines reported previously to containdeletions were analyzed using genetic markers. Each cell line revealedhomozygous deletions of at least one of the markers tested (Table 1). Nohomozygous deletions were identified using D9S161, D9S169 or D9S104. Theminimum region of overlap among these deletions was flanked by IFNA-1and D9S171. This suggested that the region between these two markerscontains a gene(s) that is involved in tumor suppression, possibly MTS.The genomic region between D9S171 and IFNA-1, particularly in thevicinity of IFNA-s, was then studied in further detail.

TABLE 1 Homozygous Deletions in Tumor Cell Lines Detected with OeneticMarkers Linked to MTS Tumor Cell Markers Lines IFNA-1 IFNA-s D9S171D9S126 D9S161 D9S169 D9S104 U-138 − − − − + + + U-118 − − − − + + + U-87− − + + + + + A-172 + − + + + + + H4 − − − − + + + NOTE: All cell linesare either gliomas or neuroblastomas available from the ATCC.

EXAMPLE 3 Genomic Clones in the MTS Region

To obtain genomic clones of the region surrounding IFNA-s, CEPH YAClibraries were screened (Cohen et al., 1993). Eleven YACs wereidentified which contained the D9S171 marker and 5 that containedIFNA-s. No YACs were isolated which included both D9S171 and IFNA-s(FIG. 2). Three of the YAC clones (C9, C6, F9) were subcloned into phageand one YAC (C6) was subcloned into a cosmid vector. These and cosmidclones provided a convenient way to produce STSs internal to knowngenetic markers and to expedite the chromosomal walk described below.

To provide an independent source of genomic DNA for construction of acontiguous genomic map of the region and to aid in production of STSs, achromosomal walk was initiated in P1 clones from IFNA-s extending towardD9S171, from D9S171 extending back toward IFNA-s, and from the YAC C6ends in both directions. A total of 27 P1 clones were isolated as partof this chromosomal walk (FIG. 2). The ordered P1s formed a contiguousassembly that stretched from IFNA-s to D9S171 with two gaps. P1 clonesas well as several phage and cosmid clones were used to generate a finestructure map of the MTS region.

EXAMPLE 4 Fine Structure Analysis of MTS Region

To construct a more detailed molecular map of the MTS region, additionalmarkers were required. DNA sequences obtained from the genomic cloneswere used to design PCR primers for STSs. These STSs served in turn tohelp order the P1 and YAC clones. A total of 54 STSs from the regionbetween IFNA-s and D9S171 were the primary basis for developing adetailed physical map of the MTS region (FIG. 2). These STS primersequences have been deposited in the Genome Database.

The set of new markers stretching from IFNA-s to D9S171 was used to test84 melanoma cell lines for homozygous deletions in the MTS region. Atotal of 52 lines revealed regions of homozygous deletion (FIG. 3).Several of the deletions were extensive; for example, 13 lines weremissing a region that included both 816.7 and 760-L.

For the purpose of localizing MTS, the most informative tumor lines fellinto two groups (FIG. 3): i) those that contained deletions of c5.1alone (class 11); and, ii) those that contained deletions of c5.3 alone(class 12). A total of 5 melanoma lines fell into these categories. Inall cases where deletions were detected, the deletion appeared to besimple; that is, there was no evidence of multiple deletion events inthe region between IFNA-s and D9S171. Together the lines harboringdeletions delineated a region of deletion overlap centered aroundmarkers c5.1 and c5.3, making the development of a complete physical mapof the region from IFNA-s to D9S171 unnecessary.

EXAMPLE 5 Identification of Cosmid c5 and P1 Colonies P1062 and P1063 asContaining MTS

A. Placement of Genetic Markers

Analysis of YAC clones and of deletions in tumor lines yielded resultsconsistent with the genetic placement of markers: IFNA, D9S171, D9S126.Three YACs contained both D9S171 and D9S126, while four YACs containedIFNA-1 and IFNA-s (FIG. 2). None contained both D9S171 and IFNA. Thissuggested that: i) IFNA-1 and IFNA-s are closely linked, and ii) D9S126and D9S171 are linked. These results were confirmed by cell linedeletions. Most cell lines that were missing D9S171 also lacked D9S126.Conversely, line U-87, although testing positive for D9S171 and D9S126,lacked IFNA-s and IFNA-1 (Table 1). One melanoma cell line, SK-MEL-5,lacked IFNA-s, D9S171 and D9S126, but not IFNA-1. Thus, IFNA-1 must bedistal to IFNA-s. Another melanoma line, SK-Mel-Zan, contained adeletion that included IFNA-1, IFNA-s and D9S171 but not D9S126, placingD9S171 between IFNA-s and D9S126. Collectively, these findings supportthe marker order given in Table 1.

The human α-interferon gene family consists of over 23 genes andpseudogenes located on chromosome 9p. This gene cluster has been clonedand sorted into 10 linkage groups (Henco et al., 1985). The linkagegroups have been partially ordered by analysis of deletion losses ofdifferent α-interferon sequences in glioma cell lines (Olopade et al.,1992). Glioma line H4 lacks both IFNA-1 and IFNA-s. It also lackssequences from linkage group IV (e.g., α13, α6 and α20). Glioma lineA172 contains both IFNA-1 and linkage group IV, but lacks sequences fromlinkage groups I (e.g., α1, α19), III (e.g., α8) and IX (e.g., α2) andIFNA-s. This analysis places IFNA-1 distal to linkage groups I, III, andIX, as well as IFNA-s. The distal boundary of the A172 deletion wasmapped within one P1 length distal of IFNA-s. Thus, linkage groups I,III, and IX must lie proximal to a point located less than 85 kb distalto IFNA-s.

B. Physical Distance Between Genetic Markers

The results did not permit a precise estimate of the distance betweenIFNA-s and D9S171, since it was not possible to isolate YACs thatcontained both markers. Furthermore, based on mapping with STSs, none offive YACs that extended distal from IFNA-s overlapped any of the 11 YACsthat extended proximal from D9S171. Given that CEPH YAC inserts averageunder 500 kb in length, the distance between IFNA-s and D9S171 is likelyto be at least this large.

The region between IFNA-s and c5.1 was covered by nine walking steps ina P1 library. Assuming that each step is on average the length of half aP1 insert, the distance between c5.1 and IFNA-s is roughly 400 kb. Thus,the tumor suppressor gene tightly linked to c5.1 which is deletedfrequently in melanoma lines must lie about 400 kb proximal to IFNA-s.

C. Deletions in Tumor Lines

Homozygous deletions of the 9p21 region were found in 57% of themelanoma tumors tested. Fourteen tumor lines contained deletions thatextended on the proximal side through 760-L, and 16 lines containeddeletions that stretched beyond 816.7 on the distal side. Assuming thedeletions are causal, that is, deletions of gene(s) in this regioncontribute to the tumor phenotype, the tumor suppressor gene(s) mustalso lie between 760-L and 816.7. The smallest deletions involvedmarkers c5.1 and c5.3. Of all of the markers tested, c5.3 was deletedfrom the largest number of lines, 51. Therefore, the most probableposition of the tumor suppressor gene(s) is very close to c5.3 becauseit is the most frequently deleted marker. Four lines contained deletionsof c5.3 alone (class 12) and one line lacked c5.1 alone (class 11). Bothof these markers were present on the same cosmid, c5. Thus, it is likelythat the tumor suppressor gene(s) includes sequences from cosmid c5. P1clones P1062 and P1063 include sequences found in c5 and surroundingcosmids. Thus, as shown further below, P1062 and P1063 contain theentire MTS region.

The results presented in the above Examples are consistent with previousgenetic studies of MTS, which found the region between IFNA-1 and D9S126to be the most probable location for MTS (Cannon-Albright et al., 1992).Recent genetic studies have confined the location of MTS further using apolymorphic (CA) repeat that lies between IFNA-s and C5.3 on P1-452(FIG. 2). Analysis of a recombinant chromosome using this marker placesMTS proximal to P1-453. Thus, MTS maps within the region wherehomozygous deletions in melanoma cell lines cluster.

These results support the view that there is a tumor suppressor locus,MTS, positioned somewhere near c5.3. All the lines that containeddeletions shared a common area of deleted DNA, with the exception of theset whose deletions were restricted to c5.1 or c5.3 (classes 11 and 12).There was no indication of non-overlapping deletions in this panel ofcell lines other than those within cosmid c5. Therefore, there is nobasis to invoke a more complex scheme involving, for example, a secondtumor suppressor locus in 9p21 distant from c5.1 and c5.3.

The observation that homozygous deletions of 9p21 occur in multipletumor types suggests that the tumor suppressor gene(s) located there maybe expressed in a wide variety of tissues. Thus, the tumor suppressorgene(s) may be similar to the p53 gene in that it may participate in thedevelopment of multiple types of cancer (see further data below). Othertypes of cancer have been reported in melanoma-prone families (Nancarrowet al., 1993; Bergman et al., 1990). A thorough deletion analysis of awide variety of tumor types using c5.1 and c5.3 (shown below) clarifiesthe importance of this tumor suppressor gene in tumors other thanmelanoma.

Some of the homozygous deletions observed remove many genetic markers.Fountain et al. reported that homozygous deletions of chromosome 9p21 intwo different melanoma lines extended 2-3 Mb (Bergman et al., 1990). Inthis study, at least one line, SK-MEL-5, contained deletions extendingfrom the most distal marker tested, IFNA-1, past D9S126, a regionapparently too large to be contained on a single YAC. The preponderanceof large deletions suggests that the region surrounding MTS is devoid ofgenes that are essential to cellular viability.

EXAMPLE 6 Isolation of MTS Candidate Genes

In the previous Examples, the results of a YAC and P1 chromosomal walkin the neighborhood of MTS were described. Fine structure-mappingexperiments with STSs derived from c5 sequences showed the presence ofsmall, non-overlapping deletions of c5 sequences in five differentmelanoma cell lines. Based on this result, it was probable that a tumorsuppressor gene, possibly MTS, lay at least partly within c5.

A further indication that c5 contained at least one gene came fromanalysis of (CpG) dinucleotide frequencies in c5 and neighboringcosmids. In mammals, virtually all housekeeping genes and nearly half ofall tissue-specific genes are associated with regions unusually rich in(CpG) dinucleotides (Bird, 1989; Larsen et al., 1992). Thus, thepresence of such “CpG islands” is indicative of genes. Cosmids c5, c12,c57, and c59 were digested with the restriction endonucleases EagI,BssHI, and SacII, enzymes whose recognition sequences include two (CpG)pairs. Only cosmids c5 and c12 contained sites for these enzymes. Cosmidc5 contained one EagI site, at least 10 BssHI sites, and at least 12SacII sites. The presence of the CpG islands in c5 and the overlappingcosmid c12 suggested that c5 indeed contained at least one candidategene for MTS.

To search for MTS, the DNA sequences of EcoRI fragments from cosmid c5were determined. When these sequences were compared against sequencesfrom GenBank, two distinct regions of c5 were identified that weresimilar to a region of a previously identified gene encoding humancyclin-dependent kinase 4 (Cdk4) inhibitor, or p16 (Serrano et al.,1993). These two genes were candidates for MTS, and were named MTS1 andMTS2. MTS1 was located near the end of cosmid c5 closest to thechromosome 9p telomere, while MTS2 was located near the centromeric endof c5. See FIG. 4B. A cosmid map showing the position of MTS1 and MTS2,as well as P1s 1062, 1063 and 1069 is shown in FIG. 4A.

A detailed comparison of genomic sequence of MTS1 from c5 with the p16mRNA sequence revealed that MTS1 contained a stretch of 307 bp that wasidentical to a portion of the p16 coding sequence. This stretch ofnucleotides in MTS1 was flanked by recognizable splice junctionsequences. Further characterization of MTS1 showed that it included theentire coding sequence of p16 plus two introns (FIGS. 5A and 5B andFIGS. 6A and 6B). Intron 1 was located 126 bp downstream from thetranslational start site; intron 2 was located 11 bp upstream from thetranslational stop site. The two introns divided the coding sequence ofMTS1 into three regions, a 5′ region of 126 bp (coding exon 1), a middleregion of 307 bp (coding exon 2), and a 3′region of 11 bp (coding exon3). SEQ ID NO:3 sets forth nucleotide sequence for the 5′ region, exon 1and part of intron 1 for MTS1. SEQ ID NO:4 sets forth the nucleotidesequence for part of intron 1, exon 2 and part of intron 2 for MTS1.

MTS2 contained a region of DNA sequence nearly identical to p16 thatextended from the 5′ end of coding exon 2 roughly 211 bp toward intron 2(FIG. 7A). However, the sequence similarity decreased until a point 51bp upstream of intron 2 in MTS1 which corresponds to the location of thefinal codon of MTS2 (FIG. 8). Comparison of sequences from MTS1 and MTS2(FIG. 8) showed that the sequence similarity between these two genesalso extended nearly 40 nucleotides upstream from the 3′ splice junctionof intron 1. Thus, portions of noncoding DNA were more conserved thansome areas of presumptive coding DNA. To exclude the possibility thatthe sequence divergence in coding DNA might be a cloning artifact, PCRprimers were designed to amplify specifically across the sequencedivergence point of MTS2. These primers amplified a fragment of thepredicted size from cosmid, P1 and genomic DNA. Therefore, the divergentsequence located near the 3′ end of exon 2 in MTS2 is a bona fidegenomic sequence. SEQ ID NO:5 sets forth the nucleotide sequence forpart of intron 1, “exon 2” and “intron 2” for MTS2. SEQ ID NO:15 setsforth the cDNA sequence for MTS2.

The occurrence of two closely related genes on cosmid c5 suggested thatother related genes might exist in this region. To test thispossibility, Southern blots were prepared from restriction enzymedigests of cosmids c5, c12, c59, P1s 1063 and 1060, and human genomicDNA. These blots were probed with a fragment containing most of exon 2from MTS1, including the region shared with MTS2. Two EcoRI fragmentswere detected with the probe in both cloned DNA and genomic DNA. Thisresult was consistent with the presence of two p16-like genes in thegenome, MTS1 and MTS2. It is also consistent with the now known presenceof MTS1E1β which is an alternate form of MTS1—containing Exons 2 and 3but not Exon 1 of MTS1.

EXAMPLE 7 Isolation and Structure of MTS1E1β

Isolation of MTS1E1β

Clones that contained MTS1E1β were isolated by hybrid selection usingthe complete MTS1 cDNA as a probe and by conventional cDNA libraryscreening. Conventional cDNA library screening was performed using aprobe derived from exon 2 of MTS1. One million clones were screened fromeach of fetal brain, normal breast and lymphocyte-derived libraries. Ahybridizing cDNA clone was isolated from the lymphocyte library. Theclone was sequenced and shown to contain E1β. It also contained exon 2(E2) and exon 3 (E3) of MTS1. Hybrid selection-derived cDNA clones wereisolated by incubating cDNA derived from ovarian tissue with cosmid c5.The cosmid was labeled with biotin and made to be single-stranded.Hybrids between c5 and the cDNA were allowed to form and then thebiotinylated cosmid was captured using streptavidin-coated magneticparticles. The selected cDNA was eluted from the cosmid, amplified byPCR, cloned and sequenced. The cDNA clones were similar to thoseisolated by library screening in that they contained E1β, E2 and E3.None of the clones contained the previously described exon 1 (see SEQ IDNO:3). The sequence for MTS1E1β cDNA is set forth in SEQ ID NO:13.

Structure of MTS1E1β

MTS1 and MTS1E1β are two forms of a single gene: the two forms bothutilize exons 2 and 3 but have different first exons. MTS1 contains thea form (E1α) which encodes the first 43 amino acids of the p16 proteinencoded by MTS1. MTS1E1β contains the β form (E1β) of exon 1. The exonstructure of the p16 gene was determined by comparing the sequence ofthe composite cDNA clones to the genomic regions from which they werederived (FIG. 13). A combination of genomic Southerns, sequence analysisof the genomic region containing P16, and long PCR were used to map thepositions of the P16 exons (FIG. 13). The p16 gene spans approximately30 kb of genomic DNA. E1β is the most 5′ of the exons, the order beingE1β, E1α, E2 and E3.

Translation of E1β in the p16 reading frame (extrapolated from thereading frame used in p16 coding exons 2 and 3) revealed an in-framestop codon positioned only 10 codons upstream of the splice junctionbetween E1β and E2. The position of the stop codon was confirmed bygenomic and cDNA sequence analysis. The first potential initiationcodon, downstream of this stop was in the p16 reading frame, immediately3′ of the E1/E2 splice junction. This potential start codon is flankedby sequences that do not closely resemble the consensus Kozak sequence(Kozak, 1987). If translated in the p16 reading frame, the E1βtranscript of the p16 gene would encode a protein of 105 amino acids.

Additional analysis of the β cDNA revealed that it possessed a large ORFin a different frame than the one used to encode p16. The ORF (referredto as ORF2) extended through E1β and continued for 67 amino acids intoE2. The entire ORF could encode a protein of 180 amino acids. However,the reading frame remained open at the 5′ end of E 1β, and therefore,may be incomplete. Statistical analysis suggested that an ORF of thissize was unlikely to occur by chance in DNA composed of random sequence(P=0.003). However, given the base composition of the β transcript, theprobability was higher (P=0.16). The predicted polypeptide was notsimilar to any previously described protein.

Identifying the evolutionarily conserved portions of E1β might provideclues as to what sequences are important for its function. Mouse p16cDNAs were isolated by a modified RACE technique called Hybrid CaptureRACE (HCR) (see Example 12) and compared to the human p16 cDNAs. Onetype of mouse P16 cDNA (the β type) possessed an exon equivalent tohuman E1β and an E2 equivalent. A second type (the a type) contained anE1α equivalent joined to E2. The E1α and E2 mouse exons were 70%identical to their human counterparts. The nucleotide sequence of themouse and human E1β exons were 51% identical (FIG. 14) and the mouse E1βexon also contained stop codons in the reading frame used to encode p16.The human and mouse polypeptides, deduced from the nucleotide sequence5′ of the stop codon, were completely divergent. Therefore, it isunlikely that the stop codons in the p16 reading frame were sequencingartifacts.

Given the uncertainty regarding the role of E1β, we analyzed thesimilarity between the mouse and human β transcripts in all threereading frames. The mouse and human β transcripts contained a large ORF(ORF 2) in a different reading frame than the one used to encode p16(ORF1). The deduced polypeptides encoded by ORF 2 were 40% identical.However, they were only 28% identical if we restricted the comparison ofthe ORF 2 peptides to the portion encoded by E1β. In contrast, the mouseand human p16 sequences were 67% identical. In addition, thepolypeptides deduced from ORF 2 contained in E2 were as similar (42%) asthe polypeptides deduced from the third reading frame in E2 (ORF 3).These results suggest that ORF 2 has not been selectively maintained andprobably does not encode a protein. The secondary structure of the humanand mouse β RNAs were also compared. No striking similarities wereidentified. Collectively, these results suggest that the β transcript isrequired for P16 function by virtue of its presence in both mouse andman; and that if it is translated, the encoded protein probablyinitiates at the first methionine in exon 2.

EXAMPLE 8 Germline Mutations in MTS1

To test whether or not MTS1 or MTS2 corresponded to the geneticsusceptibility locus MTS, genomic DNA was analyzed from eightindividuals presumed to carry MTS predisposing alleles (Cannon-Albrightet al., 1992). DNA sequences from the exons were amplified from eachsample using oligonucleotide primers (Table 2) derived from intronsequences specific for either MTS1 or MTS2.

TABLE 2 Primers for Screening Exons in MTS 1 Primers SEQ ID NO: ExonGene 1F 6 1 MTS1 1108R 7 1 MTS1 42F 8 2 MTS1 551R 9 2 MTS1 21F 10 2 MTS250R 11 2 MTS2 89F 12 2 MTS2

Exon 1 of MTS1 was amplified using primers 1F and 1108R and thensequenced using primer 1108R. Exon 2 of MTS1 was amplified using primers42F and 551R and then sequenced using primers 42F and 551R. Exon 2 ofMTS2 was amplified using primers 21F and 50R, reamplified using primers89F and 50R and then sequenced using primers 89F and 50R.

The DNA sequences of these genomic fragments revealed polymorphisms intwo of the eight individuals. The polymorphisms were not present in anyof the other samples, suggesting that they were not common in thepopulation. To demonstrate that the polymorphisms were linked to the MTSchromosome and not to the other homolog, genomic DNA from otherindividuals who carry the predisposing allele from each kindred wereanalyzed. In each case, the polymorphisms segregated with the MTSpredisposing allele. The mutation at codon 101 (gly→trp) was found in anindividual (12821) in kindred 3012. It was also found in affectedcarrier sib (13183) and in unaffected carrier cousin (14917), but not inunaffected non-carrier sib (13184). The mutation at codon 126 (val→asp)was found in an individual (15635) in kindred 1771. It was also found inaffected carrier first cousin once removed (10205), affected carrierfirst cousin (11414) and in affected carrier first cousin of 10205(10146), but not in unaffected non-carrier uncle of 10205 (10120).

The polymorphisms were single nucleotide substitutions that caused aminoacid changes (Table 3). The substitutions involved either thesubstitution of a large hydrophobic residue for small hydrophilicresidue, or the substitution of a charged amino acid for a neutral aminoacid.

TABLE 3 Predisposing Germline MTS Mutation Coding Mutation EffectLocation* G → T gly → trp 301 T → A val → asp 377 *Location of mutationin DNA sequence of SEQ ID NO:1.

Exon 2 from MTS2 showed no polymorphisms in the eight samples tested.This suggests that, at least in this set of kindreds, MTS2 does notpredispose to melanoma. It is possible that MTS2 is involved in othertypes of cancer based on its similarity to MTS1. It is also possiblethat MTS2 is a nonfunctional gene.

The finding of germline mutations in MTS1 and not in MTS2 in individualspredisposed to melanoma is consistent with the analysis of melanomahomozygous deletions.

EXAMPLE 9 Analysis of the Presence of MTS in Tumor Lines

Because of the high frequency of deletions at 9p21 in multiple tumortypes, cell lines derived from 12 different types of tumor were analyzedfor the presence or absence of MTS1. A set of sequence-tagged sites(STSs) spaced across the gene was used to test genomic DNA from tumorcell lines for the presence or absence of the expected fragment (FIGS.4A, 4B and 9). The results of this study suggested that MTS1 was deletedfrom a large percentage of tumor lines (Table 4). Homozygous deletionsoccurred in all tumor types tested other than colon and neuroblastomacell lines, the percentage of deletions varied from a low of 25% in lungcancer and leukemia to 94% in astrocytomas. In total, homozygousdeletions were detected in 135 of 290 cell lines tested. This numberyields a minimum estimate of the percentage of tumor lines that harbordeletions because the STSs used for the analysis did not cover theentire gene. Thus, certain small deletions could have escaped detection.In addition, lesions such as insertions or deletions of a fewnucleotides, and nucleotide substitutions, would be missed by thisapproach.

TABLE 4 Homozygous Deletions in Tumor By Tumor Type No. No. % Tumor TypeLines Deletions Deletions melanoma 99 57 58 leukemia 4 1 25 lung 59 1525 neuroblastoma 10 0 0 bladder 15 5 33 renal 9 5 56 astrocytoma 17 1694 colon 20 0 0 breast 10 6 60 ovary 7 2 29 glioma 35 25 71 osteosarcoma5 3 60 TOTAL 290 135 47%

To improve the estimate of the total number of cell lines containingMTS1 mutations, 34 of the cell lines that did not suffer obvioushomozygous deletions of MTS1 sequence were examined more closely forlesions in MTS1. Sequences comprising nearly 97% of the MTS1 codingsequence were amplified and screened for polymorphisms. Eighteen somaticmutations in exon 2 or exon 1 of MTS1, distributed in 14 out of 34melanomas, were observed (Table 5). Three of these mutations wereframeshifts, 7 were nonsense mutations, 4 were missense mutations and 4were silent. Three of the 4 lines that contained silent mutations alsocontained additional mutations and 16 of 18 mutations were located incoding exon 2. All but one line contained exclusively hemi- orhomozygous polymorphisms, suggesting that the other homologouschromosomes had incurred deletions. The single line that washeterozygous contained two different nonsilent mutations, a findingconsistent with the view that each homolog had undergone independentmutational events. Based on this DNA sequence and deletion analysis ofMTS1, a minimum of 75% of melanoma lines contained mutant MTS1 or hadlost the gene from both homologs.

TABLE 5 Somatic MTS Mutations in Tumors Cell Coding Lne Mutation EffectLocation* SK-M-ste G → A none 264 G → A gly → ser 265 SK-M-swi C → T arg→ stop 172 SK-M-ris G → A ala → thr 442 SK-M-beh 5 base deletionframeshift 290-294 SK-M-178 C → T arg → stop 238 SK-M-sta G → A trp →stop 330 SK-M-uti C → T arg → stop 238 SK-M-EML131 8 base deletionframeshift 172-179 C → A none 177 SK-M-koz(het.**) C → T pro → leu 341 C→ T none 237 C → T arg → stop 239 SK-M-kra C → T pro → 1eu 341 SK-M-kuuC → T none 378 SK-M-mar G → A trp → stop 329 SK-M-whi C → T gln → stop148 SK-M-adl(het.) 2 base deletion frameshift 128-129 *Location ofmutation in DNA sequence of SEQ ID NO:1. **Het. stands for“heterozygote” and refers to the presence in the sample of both wildtypeand mutant sequences.

The preponderance of lesions in MTS1 (deletions and nucleotidesubstitutions) indicates that MTS1 or a closely linked locus contributesto the tumor phenotype. Cells that suffer these lesions enjoy aselective advantage over cells that do not. The alternative explanation,that the lesions are random events that have nothing to do with cellgrowth, is unlikely for several reasons. First, the high correlationbetween tumor phenotype and mutation at MTS1 implies a causal relationbetween MTS1 mutations and tumor formation. Second, MTS1 influencessusceptibility to melanoma, and thus is implicated independently as atumor suppressor gene. Third, the biochemical function of p16 as apotent inhibitor of a Cdk neatly fits a model where p16 acts in vivo asa general inhibitor of the onset of DNA replication.

It is possible that mutation or loss of MTS 1 is a product of cellgrowth in culture. However, a high percentage of primary leukemia cellsalso contain homozygous deletions of the α-interferon gene cluster, agene family located less than 500 kb from MTS1 (Diaz et al., 1990).Previous deletion studies suggest that deletions of α-interferon genesinvariably involve markers that extend beyond MTS1 toward the centromere(Weaver-Feldhaus et al., 1994). Because homozygous deletions of the MTS1region occur in primary tumor cells as well as cultured cell lines, thedeletions observed in tumor cell lines are unlikely to be purely anartifact of cell growth in culture. Nevertheless, the question of whenMTS 1 mutations occur during the progression of tumors will be answeredbest by analysis of primary tumor samples.

The Role of MTS1 in vivo

In all eukaryotic cells, cell division requires passage through twocritical decision points: the G1 to S transition, where DNA synthesiscommences, and the G2 to M transition, where mitosis begins. In mammals,the machinery that controls cell division has multiple components, manyof which are related (for review see Sherr, 1993). The Cdks may be atthe heart of the control apparatus, in that they regulate byphosphorylation a number of key substrates that in turn trigger thetransition from G1 to S and from G2 to M. The G1 to S transition isperhaps the more critical decision point, as it occurs first in the cellcycle. So far, four types of Cdk have been defined (Cdk2-5) that mayparticipate in G1 to S control, as well as a set of positive regulatorsof these Cdks (cyclins C, D1-3, E). Recently several negative regulatorshave also been identified, including p16, p15, p18, p20, p21, and p27(Xiong et al., 1993; Serrano et al., 1993; Gu et al. 1993; El-Diery etal., 1993; Harper et al., 1993; Hannon and Beach, 1994; Polyak et al.,1994b; Toyoshima and Hunter, 1994; Guan et al., 1995). These negativeregulators appear to act by inhibiting the kinase activity of the CDKs.Some of the cell cycle regulators are involved in human cancers (forreview, see Hunter and Pines, 1994). p20 inhibits Cdk2 and possiblyother Cdks while p16 (also called MTS1, CDKN2, or INK4a) inhibits Cdk4but apparently does not inhibit Cdk2 in an in vitro assay (Serrano etal., 1993). Based on in vitro studies and on its interaction with p53,p21 has been proposed as a general inhibitor of all Cdks (Xiong et al.,1993). Thus, in vitro, p16 appears more specific than p21. Each of theseinhibitors is expected to antagonize entry into S phase. Also, cyclin D1or CDK4 is overexpressed in some breast carcinomas and the p16 gene ismutated or deleted in a large number of cell lines and primary tumors(Buckley et al., 1993; Caldas et al., 1994; Kamb et al., 1994b; Mori etal., 1994; Tam et al., 1994a). These results suggest that certaincyclins and CDKs are protooncogenes and that P16 (MTS1) is a tumorsuppressor gene. The biochemical behavior of p15, p18, p21 and p27indicate that they too may be tumor suppressors, but detailed mutationalanalysis of their genes in tumors or cell lines has not been reported.The results presented here provide evidence that MTS1 functions in vivoas an inhibitor of cell division.

The p16 gene (MTS1), located in the 9p21 segment of human chromosome 9,is especially interesting because it is mutated or homozygously deletedin a high percentage of some types of tumors and tumor-derived celllines (Caldas et al., 1994; Kamb et al., 1994b; Mori et al., 1994;Nobori et al., 1994). In addition, MTS1 mutations segregate withpredisposition to melanoma in several kindreds known to carry 9p21linked melanoma susceptibility (Hussussian et al., 1994; Kamb et al.,1994a). However, there are unresolved questions regarding the role ofMTS1 in hereditary and sporadic cancer. Several melanoma-prone kindredswith high LOD scores for 9p21 markers do not reveal mutations in MTS1coding sequences. Also, the preponderance of MTS1 homozygous deletionsin tumors and cell lines is atypical for tumor suppressor geneinactivation and may imply the presence of another gene(s) near MTS1which also participates in cancer formation.

Recent reports suggest that some mitogenic and antimitogenic signalsaffect cell cycle progression, at least in part by regulating theactivity of CDK inhibitors (Firpo et al., 1994; Hannon and Beach, 1994;Kato et al., 1994; Polyak et al., 1994a; Slingerland et al., 1994). Forexample, TGFβ-induced cell cycle arrest may be mediated by activation ofp15 and p27. Conversely, p27 may be negatively regulated duringIL-2-induced mitogenic activation of quiescent T lymphocytes.Comparatively little is known about the regulation of MTS1. Many recentreports provide evidence that MTS1 levels may be regulated in part by Rbprotein (Serrano et al., 1993; Li et al., 1994a; Tam et al., 1994b;Parry et al., 1995). These and other findings (Serrano et al., 1995)have contributed to a model for MTS1 action in which MTS1 inhibitsCDK4/6 and thereby prevents phosphorylation of Rb. Rb in turnparticipates in a feedback loop to limit the levels of MTS1.

These results provide genetic evidence for a pre-eminent role of MTS1 incontrol of the cell cycle. Moreover, the results suggest that the targetof MTS1 in vivo is a major factor in tumorigenesis. If MTS1 inhibitsCdk4 in vivo and not Cdk2, Cdk4 may be a strong candidate for anoncogene. The prevalence of mutations in the MTS1 gene implies that Cdk4may serve as a general activator of cell division in most, if not all,cells. Further biochemical studies of the effects of MTS1 on differentCdks may help clarify the hierarchy of Cdk activity in both normal cellsand transformed cells. By analogy with p16, if p21 acts as a generalinhibitor of Cdks, its gene may also be lost or mutated in a largepercentage of tumors.

If MTS1 is a general tumor suppressor active in most normal cells,germline mutations in MTS1 might be expected to predispose to cancersother than melanoma. For example, germline mutations in the p53 genesuch as those found in Li-Fraumeni syndrome increase the likelihood ofmany tumor types including childhood sarcomas, breast cancer (Malkin etal., 1990). Previous studies have found an unusually high incidence ofpancreatic cancer in some families that are prone to melanoma (Bergmanet al., 1990; Nancarrow et al., 1993). This observation accords with thefinding that homozygous deletions of MTS1 occur in pancreatic tumorlines. It is possible that the genetics of MTS1 predisposition may bedifferent from the somatic cell genetics of MTS1. For instance, largedeletions that remove many kilobases of DNA from the region surroundingMTS1 may be lost from the human gene pool, due to a selectivedisadvantage. However, such deletions may be favored in transformedsomatic cells, perhaps because they remove multiple genes. Thispossibility is consistent with the existence of a second gene withstriking similarity to MTS1, called MTS2. MTS2 is located roughly 12 kbupstream of exon 1 of MTS1, the first exon of MTS2 being roughly 2.5 kbupstream of the second exon of MTS2. MTS2 may function in a fashionsimilar to MTS1. Deletions that remove both MTS1 and MTS2 might confer agreater growth advantage to cells than mutations that inactivate eithergene alone. Alternatively, the two different genes may function in anon-overlapping or partly overlapping set of cell types. Thesepossibilities remain to be thoroughly explored.

EXAMPLE 10 Mutational Analysis of MTS1E1β

Both the preponderance of homozygous deletions which inactivate P16 intumor derived cell lines, and the 9p21-linked melanoma-prone kindredsthat do not reveal mutations in P16 have led others to propose thepresence of another gene(s) near P16 which is also involved in cancerformation (Cairns et al., 1994; Spruck et al., 1994). If E1β encoded aprotein which was involved in regulating cell growth, then thesesequences could contain mutations in either sporadic and/or familialcancer that would have been missed in earlier studies. Therefore, E1βwas screened for mutations in cell lines derived from various tumors andin some melanoma prone kindreds.

Genetic characterization of the melanoma-prone pedigrees has beenpreviously reported (Cannon-Albright et al., 1992). Isolation of genomicDNA from melanoma prone kindreds (Kamb et al., 1994a) and from celllines (Liu et al., 1995) has been previously described. PCRamplification for E1β was performed using the forward primer(5′-AGTCTGCAGTTAAGG-3′ SEQ ID NO:33) and the reverse primer(5′-GGCTAGAGGCGAATTATCTGT-3′ SEQ ID NO:34) for 30 cycles using thefollowing conditions: 97° C. for 3 seconds, 65° C. for 10 seconds, 75°C. for 20 seconds. The amplification reactions were diluted 100 fold andamplified again under the same reaction conditions with the same forwardprimer and the reverse primer (5′-CACCAAACAAAACAAGTGCCG-3′ SEQ IDNO:35). PCR products were run on a 1% agarose gel and were extractedusing Qiagen beads (Qiagen, Inc.). The products were sequenced using theCyclist Sequencing kit (Stratagene) with the forward primer mentionedabove (SEQ ID NO:33).

No sequence variants of E1β were detected in a set of 24 cell linesderived from 4 tumor types (Table 6) or in 6 melanoma kindreds withsignificant haplotype sharing among affected family members(Cannon-Albright et al., 1992), but which did not reveal P16 mutationsin a previous study (Kamb et al., 1994a). These experiments suggest thatmutations in E1β are not a common event during tumor progression, norare they responsible for 9p21-linked melanoma susceptibility in thesekindreds.

TABLE 6 Cell Lines Screened for E1β Mutations Type Number lung 3 bladder7 glioma 9 melanoma 5 total¹ 24 ¹These cell lines were previously shownnot to contain homozygous deletions in the P16 region or harbor P16coding sequence mutations (Liu et al., 1995). Based on previous results(Liu et al., 1995), a similar number and type of cell lines would havecontained 4 point mutations in the p16 coding sequence, confined to thebladder and melanoma groups.

EXAMPLE 11 Mutation Screening of MTS2

MTS2 Mutation Screening in Cell Lines

The preponderance of homozygous deletions that remove MTS1 in tumorderived cell lines may suggest the presence of another gene or genesnear MTS1 which are also involved in cancer formation. If the MTS2 genewere involved in sporadic cancer, it might contain mutations in celllines of tumor origin. Therefore, MTS2 coding sequences were screenedfor mutations in a set of tumor cell lines.

PCR amplification for exons 1 and 2 of MTS2 were performed as describedin Kamb et al. (1994a). The primer pair 2E1.F1(5′-AGGGAAGAGTGTCGTTAAG-3′ SEQ ID NO:19) and 2E1.R2(5′-AGACTCCTGTACAAATCTAC-3′SEQ ID NO:20) was used to obtain exon 1.Primer pair 89F (SEQ ID NO:12) and 50R (SEQ ID NO:11) was used to obtainexon 2. After amplification, the DNA products were run on a 1% agarosegel and were extracted using Qiagen beads (Qiagen, Inc.). The productswere sequenced using the Cyclist Sequencing kit (Stratagene) with primer2E1.F1 for exon 1 and 89F and 50R for exon 2.

MTS2 coding sequences were screened for mutations in a set of cell linesderived from bladder, glioma, astrocytoma, lung, renal, and melanomatumors. All these cell line types contain homozygous deletions of MTS2and MTS1 at high frequencies (Kamb et al., 1994b). Cell lines derivedfrom melanoma, lung, renal, and bladder carcinoma have been shown tocontain point or frameshift mutations in MTS1 (Liu et al., 1995). Gliomaand astrocytoma cell lines, however, have not been shown to contain suchMTS2 mutations. The particular cell lines used in these screeningexperiments were selected from a group shown previously not to harborhomozygous deletions of MTS2 and MTS1 sequences (Kamb et al., 1994b).

No MTS2 mutations were found in MTS2 coding sequences in any of the 58cell lines that were screened (see Table 7). Based on previous studiesof MTS1 in these cell line types, the set would be expected to containabout 8 MTS1 mutations confined to the bladder, melanoma, lung, andrenal group (Liu et al., 1995). Thus, no evidence for somatic mutationsin MTS2 was obtained from this set of tumor cell lines.

TABLE 7 Mutation Screening of MTS2 in Cell Lines Polymorphisms¹ # # ofCoding Cell Line Type Screened Changes Type of Change Effect Astrocytoma2 0 Bladder 4 1 G → A; C → A None Glioma 6 0 Melanoma 17 2 G → A: C → ANone Renal 4 1 G → A; C → A None Lung 10 1 G → A; C → A None Small CellLung 7 2 G → A; C → A None Non-small Cell 8 0 Lung Total 58 7 ¹Thesecommon polymorphisms (Kamb et al., 1994a) are located in intron 1 nearthe 3′ acceptor site at nucleotide positions −27 (C to A) and −103 (G toA).

MTS2 Mutation Screening in Kindreds

The possibility that the MTS2 gene accounts for the melanomasusceptibility in the 9p21-linked, melanoma-prone kindreds that do nothave MTS1 coding sequence mutations is attractive. Geneticcharacterization of the melanoma-prone pedigrees has been previouslyreported (Cannon-Albright et al., 1992). Genomic DNA from family memberswas isolated from lymphocytes which had been separated from whole bloodusing standard procedures (Kamb et al., 1994a). Screening was performedas described above for mutations in MTS2 coding sequences in 6 kindredswith high LOD scores for 9p21-linked predisposition to melanoma butwhich did not reveal MTS1 mutations in a previous study (see Table 8)(Kamb et al., 1994a). No mutations in MTS2 were detected. Theseexperiments thus provide no evidence that MTS2 lesions contribute tohereditary melanoma although such a possibility cannot be ruled outsimply based on these limited experiments.

TABLE 8 Melanoma-prone Kindreds Screened for MTS2 germline MutationsCases with Kindred LOD Score Total Cases Haplotype 3346 5.97 21 21 31371.9 17 21 1764 1.04 4 4 3006 0.19 6 3 3161 −0.01 10 8 3343 −0.53 10 8

EXAMPLE 12 Expression of MTS1 and MTS1E1β RNAs

Two P16 Promoters

The two different forms of the P16 mRNA could be generated in twopossible ways. Transcription could initiate from different promoters, orthe mRNA could be derived from a single promoter and then alternativelyspliced to generate the different forms of the transcript.

Evidence for separate α transcript and β transcript promoters wasobtained by demonstrating that the α form was transcribed in cell lineseven when the upstream E1β sequences were deleted. Cell lines A375 andSK-mel 93 contain a deletion with one breakpoint between E1α and E1β(FIG. 13). The proximal breakpoint has not been precisely mapped ineither cell line, but was at least 85 kb upstream of the 5′ end of E1β.Using RT-PCR with α-specific primers, both of these cell lines wereshown to express the α transcript (FIG. 15). The procedure for theRT-PCR is as follows: cDNA was synthesized from total RNA (Sambrook etal., 1989) isolated from T cells, cell lines, or human tissues(Clontech). The cDNA reactions employed random 9 mers to prime DNAsynthesis and Superscript II reverse transcriptase (Bethesda ResearchLaboratories). cDNA yields were calculated by including α³²P-dATP(Amersham) in the synthesis reaction (0.1 Ci/mmole) and determining theamount of radioactive nucleotide incorporated into the final product.P16 α and P16 β transcript levels were analyzed by PCR using α or βspecific forward primers and heminested reverse primers from E2 in twosuccessive rounds of amplification. In the initial amplification, 2 ngof cDNA was amplified with the α-specific primer AS.1(5′-CAACGCACCGAATAGTTACG-3′ SEQ ID NO:26) or the β-specific primer BS.1(5′-TACTGAGGAGCCAGCGTCTA-3′ SEQ ID NO:27) and X2.R140′(5′-AGCACCACCAGCGTGTC-3′ SEQ ID NO:22). The reactions were done on aPerkin-Elmer 9600 thermal cycler for 20 cycles under the followingconditions: 97° C. for 3 seconds; 65° C. for 10 seconds; 75° C. for 20seconds. These reactions were diluted 100 fold and reamplified with AS.1or BS.1 and X2B (5′-CGTGTCCAGGAAGCCC-3′ SEQ ID NO:23). The X2B oligo wasradiolabeled at its 5′ end (Sambrook et al., 1989) with γ³²P-dATP(DuPont). PCR conditions were as above, but for only 15 cycles. Toeliminate problems due to genomic DNA contamination, the PCR productsspanned the E1α or E1β/E2 splice junction. The products were resolved byelectrophoresis through a denaturing 5% polyacrylamide gel. Dried gelswere exposed to X-OMAT (Kodak) film overnight.

The results suggest that the α transcript initiates from a promoter thatis independent of sequences 5′ of E1β. An alternative explanation isthat the deletions fused ectopic promoter sequences to E1α. However,this seems unlikely given that A375 and SK-mel 93 are independentlyisolated cell lines. The exact location of the α promoter is not clear,but RNase protection analysis indicated that it initiated at least 440bp upstream of the p16 initiation codon. Thus, the human p16 gene iscomplex, with two partially overlapping transcripts with distinct codingpotential, produced from separate promoters, P_(α) and P_(β).

Expression Pattern of P16

Clues to the function of genes may emerge from analysis of theirexpression pattern in different tissues. To determine the expressionpattern of P16, a set of cDNA samples prepared from eleven tissues werescreened by PCR with α and β specific primers (FIGS. 16A-D). Both formsof P16 transcript were detected in all tissues examined, though therewere some differences. For example, in spleen the ratio between the αand β forms was skewed toward β. In contrast, the ratio in breastfavored α. These expression data are consistent with studies which founddeletions and point mutations of P16 in cell lines derived from manydifferent tissue types (Kamb et al., 1994b; Liu et al., 1995) in thatthey suggest roles for p16 in multiple tissues.

Given the biochemical function of p16, demonstrated in vitro to be aninhibitor of CDK4 and CDK6 (Serrano et al., 1993; Li et al., 1994a;Parry et al., 1995), the expression of P16 was analyzed as cellstraversed the cell cycle. Human peripheral blood lymphocytes (PBLs) werestimulated by phytohemaglutinin (PHA) plus interleukin-2 (IL-2), andcells were harvested at different times after stimulation. These cellswere analyzed by flow cytometry to determine their cell cycle stage, byRT-PCR to determine the relative levels of P16 gene expression, and byWestern blot to determine the levels of p16 protein. The peripheralblood lymphocytes were isolated from blood drawn from normal adultdonors and partially purified by floatation on Ficoll-Hypaque gradients(Boyum, 1968). The lymphocytes were further purified by counter currentelutration as peviously described (Elstad et al., 1988). These authorsestimated that a cell population, prepared in this manner, was 98% pureB and T cells. The purified cells were grown in RPMI (Gibco)supplemented with 10% fetal bovine serum. Quiescent cells we;e inducedby 10 μg/ml PHA (Sigma) and 10 U/ml IL-2 (Sigma). Cell cycle progressionwas monitored by flow cytometry. RNA was isolated from primary T cellsusing RNazol B (CINNA/BIOTECX Laboratories, Inc.) as described by themanufacturer. The quantitative behavior of the RT-PCR was confirmed bycreating serial dilutions from the T cell cDNA isolated after induction.The amount of target cDNA present in the undiluted sample was quantifiedby determining the dilution value at which the target was no longeramplifiable. Although the results from the different PCR experimentswere in agreement, the dilution experiments suggested that we could onlydetect changes in RNA levels if they were greater than 4 fold. The cDNAsamples from the Rb⁺ and Rb⁻ cell lines were also analyzed in thismanner. Human actin was easily detected and present in similar amountsfrom each cDNA sample (whether from tissues, cell lines, or T cells).

The ratio of the two forms of P16 transcript changed dramaticallythrough the cell cycle (FIGS. 16B-C). Initially, the β form was low, butby 30 to 40 hours after stimulation, the level began to rise. Duringthis time, the expression level of the α form remained relativelyconstant, perhaps increasing slightly. By flow cytometry, the ratiochange was correlated with cells exiting G_(o) and entering S phase. Thequantitative behavior of the RT-PCR was examined by template dilutionexperiments. Based on those experiments, RT-PCR was sensitive to fourfold or greater changes in transcript level. The β induction wasestimated to be at least ten fold. Therefore, as T-cells entered thecell cycle they altered the relative amounts of the two forms of the P16transcript so that the ratio changed in the favor of β.

We also examined the level of p16 protein expression as the T-cellstraversed the cell cycle. Protein was isolated from the cells at varioustimes after mitogenic induction, and the isolated protein was subjectedto Western analysis. The levels of p16 protein were determined using ap16 antibody raised against the 20 C-terminal amino acids of thecomplete polypeptide. As the cells exited Go, the level of p16 proteinremained relatively constant. Thus, both the p16-encoding RNA (the αtranscript) and p16 protein remained relatively constant during the cellcycle. Others have reported a moderate increase in p16 levels during Sphase (Tam et al., 1994b). We did not see an accumulation of p16, whichmight reflect differences in p16 regulation in different cell types, orreflect problems in detecting a two to three fold increase in protein(or cDNA) levels.

Expression of P16 in Tumor Cell Lines

Previous studies have suggested that Rb influences the expression of p16(Serrano et al., 1993; Li et al., 1994a; Parry et al., 1995). We testedthe effect of the Rb status of cells on the expression of the β mRNA(FIG. 16D). cDNA was prepared from a set of cell lines, five of whichcontained wild type Rb protein, and six of which containednon-functional Rb protein (Parry et al., 1995). As expected, αtranscript was only detected in Rb-negative lines. However, the βtranscript was present in both Rb-positive and Rb-negative cell lines.Therefore, in contrast to α, expression of the β RNA is independent ofthe mutation state of Rb in tumor-derived cell lines.

There is evidence that p16 is a member of a multigene family (Guan etal., 1995). By analogy with other multigene families, the members ofthis family might carry out redundant functions, different functions, orfunction in different temporal or tissue-specific patterns. Therefore,given the low level of p16 protein and apparent lack of P16 regulationby Rb, it is possible that P16 does not regulate the cell cycle in Tlymphocytes. However, because the β transcript was dramatically inducedupon T cell induction, and because P16 is deleted in a high percentageof T cell-derived tumors (Hebert et al., 1994), it seems likely that p16carries out an important function in human T cells. A dramatic effect ofRb on p16 has only been observed in virally transformed or tumor-derivedcell lines. Perhaps P16 is regulated in some other manner in wild typetissue.

E1β is a Conserved and Regulated Part of p16

Although the role of the β transcript is unclear, the results suggestthat it is important for the function of the p16 locus because: (i) E1βis conserved in mice; (ii) the relative amount of the β transcript isregulated in both a tissue-specific and cell-cycle dependent manner; and(iii) two cell lines harbor homozygous deletions that remove E1β, butnot E1α. These results suggest that E1β is required for wild-type P16function.

The mouse β cDNA was isolated and compared to MTS1E1β of humans. MousecDNA clones were isolated by a modified hybrid selection procedurecalled hybrid capture RACE (HCR). Mouse polyA⁺-enriched RNA was isolatedfrom breast and thymus tissues. First strand cDNA synthesis reactions(Sambrook et al., 1989) employed random 12 mers and Superscript IIreverse transcriptase (Bethesda Research Laboratories). After secondstrand synthesis, the cDNAs were “anchored” by ligation of a specificdouble stranded oligo (dsRP.2) (5′-TGAGTAGAATTCTAACGGCCGTCATTGTTC-3′ SEQID NO:28) to their 5′ ends. The 5′ end of the second cDNA strand was theonly phosphorylated DNA end in the ligation reaction. After the ligationthe anchored cDNA was purified by fractionation on Sepharose CL-4Bcolumns. The anchored cDNA was amplified with a P16 specific reverseprimer (5′-AGCGTGTCCAGGAAGCCTTC-3′ SEQ ID NO: 29) and a nested versionof RP.2 (RP.B) (5′-TGAGTAGAATTCTAACGGCCGTCATTG-3′ SEQ ID NO:30) followedby capture with a biotinylated gene-specific oligonucleotide(5′-ACTGCGAGGACCCCACTACCTTCTCC-3′ SEQ ID NO:31) upstream of the reverseprimer used in the first amplification. The captured cDNAs wereamplified again, using RP.B and a gene-specific reverse primer(5′-GAACGTTGCCCATCATCATC-3′ SEQ ID NO:32) upstream of the capture oligo.The resultant products were gel purified, cloned, and sequenced. Thesequence for the mouse P16 oligonucleotides was determined by cloningand sequencing a mouse genomic clone that contained sequenceshybridizing to a human E2 probe at low stringency.

Comparison of the mouse β transcript to the human suggests that E1β doesnot encode a protein. Only the sequence comprising the p16 reading framein E2 was rigorously conserved. Therefore, if the β transcript weretranslated, it seems likely that the protein would initiate in E2 and betranslated in the same frame used to encode p16. The deduced polypeptidewould have a calculated molecular weight of 10 kDa and retain 2¾ of the4 ankyrin repeats present in p16. However, p15 contains only 3½ ankyrinrepeats (Hannon and Beach, 1994), and other proteins fold and functionwith only one or two repeats. Whether a p10 molecule exists in vivo andwhether it inhibits CDK4/6 remain to be tested.

Function of the β RNA

If the role of the β transcript were to inhibit cell growth, we mightfind mutations which disrupt E1β in tumor-derived cell lines. Consistentwith this view are two melanoma cell lines with deletions that removeE1β yet continue to express the α transcript. The p16 coding sequence iswild type in these cell lines. Nevertheless, no small genetic lesions inE1β (e.g. base substitutions) were found in a set of 25 tumor celllines. Therefore, it is difficult to conclude that E1β was the target ofthe homozygous deletions. If the E1β exon does not encode a protein,small genetic lesions may be insufficient to disrupt its function.Alternatively, the target of the deletions mentioned above might havebeen some other gene. For example, it is possible that the p15 gene(MTS2) was the relevant target of the deletions in these melanoma celllines. In that view, E1β was deleted simply because it is closer to P15than is E1α. However, since we were unable to detect P15 point mutationsin a variety of cell lines, and because there were no cell lines thatcontained deletions which specifically removed P15 (Kamb et al., 1994b),this explanation seems unlikely.

The genetic evidence suggests that p16 and Rb are members of a growthregulatory pathway that is often inactivated during tumor progression.If the role of the β transcript is to negatively regulate cell growth,perhaps it is part of another pathway which must be mutatedindependently from p16 and Rb. This would explain why deletions whichspecifically disrupt E1β have only been seen in Rb cell lines. Based onits expression pattern, it seems likely that E1β plays a role inactively cycling cells. A definitive conclusion on the role of E1βawaits analysis of its expression in vivo.

EXAMPLE 13 Expression of MTS2 mRNA

RNA was isolated from cell lines or from primary T cells using RNazol B(CINNA/BIOTECX Laboratories, Inc.) as described by the manufacturer.cDNA was synthesized from total RNA (Sambrook et al., 1989) using arandom 9 mer to prime DNA synthesis. cDNA yields were calculated byincluding α³²P-dATP (Amersham) in the synthesis reaction (0.1 Ci/mmole)and determining the amount of radioactive nucleotide incorporated intothe final product. MTS2 expression was analyzed by PCR using heminestedreverse primers in two successive rounds of amplification. In theinitial amplification, 2 ng of cDNA was amplified with E1F(5′-TGAGGGTCTGGCCAGC-3′ SEQ ID NO:21) and X2.R140′(5′-AGCACCACCAGCGTGTC-3′ SEQ ID NO:22). The reactions were done on aPerkin-Elmer 9600 thermal cycler for 20 cycles under the followingconditions: 97° C., 3 seconds; 65° C., 10 seconds; 75° C., 20 seconds.These reactions were diluted 100 fold and reamplified with E1F and X2B(5′-CGTGTCCAGGAAGCCC-3′ SEQ ID NO:23). The X2B oligo was radiolabeled atits 5′ end (Sambrook et al., 1989) with γ³²P-dATP (DuPont). PCRconditions were as above, but for only 15 cycles. The resultant productswere resolved by electrophoresis through a denaturing 5% polyacrylamidegel. Dried gels were exposed to X-OMAT (Kodak) film overnight.

MTS2 Expression in Different Tissues

MTS2 was found to be expressed in many tissue types, including thosethat give rise to tumors in which MTS2 is homozygously deleted (see FIG.10A). However, there were some differences among the tissues. Forexample, while the MTS2 transcript was easily detected in lung tissue,it was undetectable in prostrate and brain tissue. In contrast,expression of the closely related MTS1 gene was detected in all of thetissues examined. It is unknown if the tissue specific differences inMTS2 RNA levels reflects a tissue specific requirement for the MTS2protein.

MTS2 Expression Throughout the Cell Cycle

If MTS2 regulates important transitions in the cell cycle, itsexpression might vary through the cell cycle. For instance, in normaldividing cells the abundance of p21 mRNA varies as a function of cellcycle phase (Li et al., 1994b). To test whether or not MTS2transcription was regulated through the cell cycle, quiescent human Tcells were stimulated with PHA and IL2 and monitored at various stagesafter stimulation (see FIG. 10B). No obvious trend in MTS2 expressionlevel was detected as the cells exited G_(o) and passed through the cellcycle phases. In contrast, the expression of a control gene, CDK4, didchange as expected (Matsushime et al., 1992). Thus, no evidence wasfound for the differential expression of MTS2 mRNA through the cycle ofnormally dividing cells.

The MTS1 protein has been proposed to participate in a growth regulatorypathway involving the retinoblastoma protein Rb (Serrano et al., 1993;Guan et al., 1995; Serrano et al., 1995). Recent work has providedstrong circumstantial evidence for the view that expression of MTS1 iscontrolled, at least in part, by Rb (Li et al., 1994a; Parry et al.,1995). The biochemical similarities between MTS1 and MTS2 suggest thatMTS2 might also be regulated by Rb. This possibility was tested bycomparing levels of MTS2 mRNA in Rb positive cell lines and Rb negativecell lines. No correlation between Rb status and MTS2 RNA levels wasdetected (see FIG. 10C). This suggests that the Rb status of the cellline does not dramatically affect the abundance of MTS2 transcript.Thus, in contrast to MTS1, MTS2 expression may be independent of Rb.

EXAMPLE 14 Ectopic Expression of MTS1 and MTS2

A 483 bp fragment of MTS1 was generated by a polymerase chain reactionusing primers MTS1.F (5′ AAA GGA TCC ATT GCC ACC ATG GAG CCG GCG GCG GGGAGC AGC ATG GAG CCT TCG GCT 3′) (SEQ ID NO:17) and E3.R (5′ TTT GAA TTCAAT CGG GGA TGT CTG 3′) (SEQ ID NO:18). Primer MTS1.F was designed toinclude restriction enzyme sites near the 5′ end for future cloning anda Kozak consensus sequence (Kozak, 1987). Template DNA for this reactionwas cDNA from breast tissue. This generated fragment was inserted intothe expression vector pcDNA3 (In Vitrogen) which had been digested withEcoRI and BamHI. pcDNA3 contains a cytomegalovirus (CMV) promoter andcodes for resistance to ampicillin and neomycin. The resultingrecombinant vector, pcDNAp16, was then inserted by electroporation intocell line HS294T. HS294T is derived from a melanoma and contains ahomozygous deletion of both MTS1 and MTS2. HS294T was grown in DMEM(Gibco) supplemented with 10% fetal bovine serum, non-essential aminoacids, sodium pyruvate, and L-glutamate. The cells were grown at 37° C.in 5% CO₂. HS294T was cotransformed with a 1:4 ratio of pSS(Stratagene), which confers hygromycin resistance to transformed cells,and either pcDNA3 (Invitrogen) expression vector containing the MTS1coding sequence inserted downstream of the CMV promoter, or the pcDNA3vector without an insert.

The coding portion of MTS2 was similarly cloned into pcDNA3, againforming a Kozak sequence, to yield pcDNAp15 and was inserted intoHS294T. For this, MTS2 cDNA as prepared in Example 13 above was used.

Plasmid pSS (Stratagene) which contains the selectable marker forhygromycin resistance was simultaneously cotransferred with the pcDNAp16or pcDNAp15, the pSS being present in the electroporation at 20 μg percuvette. The conditions for electroporation were 800 μL of cells percuvette at 1.5×10⁶ cells/ml and 500 μF capacitance, 400 volts. Controlexperiments were performed using pcDNA3 plus pSS. The electroporatedcells (about 400 μL) were placed in petri dishes with 300 μg/mLhygromycin. The number of colonies (foci) were counted after 14 days.The results are shown in Table 9.

TABLE 9 pcDNAp15 pcDNAp16 Plasmids Colonies/plate Colonies/platepcDNA3 + pSS 26.6 ± 4.8 17.2 ± 0.15 pcDNAp15 + pSS 3.8 ± 0.8 —pcDNAp16 + pSS — 1.1 ± 0.5

When a construct containing the entire MTS2 coding sequence fused to theCMV promoter was transformed into HS294T, it inhibited colony formationby a factor of seven when compared to controls, comparable to the effectof ectopic expression of MTS1. This result indicates that ectopicexpression of MTS2 is sufficient to inhibit cell growth. It is not clearwhether the transformed cells are arrested in G1, as seems to resultfrom ectopic expression of MTS1, or growth is arrested in some othermanner. It can be concluded from the data of Table 9 that overexpressionof p15 or p16 in a cell line which otherwise would lack p15 or p16expression (here because of a homozygous deletion) inhibits growth ofthe cells. The precise mechanism is unclear but possibilities are thatthe p15 or p16 overexpression stops cell division or kills the cell.These results suggest that p15 and p16 function in vivo as bona fidetumor suppressor proteins and therefore p15 and p16 possibly havetherapeutic uses.

For many reasons MTS2 is an attractive candidate for a tumor suppressorgene. It possesses extensive sequence similarity to MTS1, it binds toand inhibits CDK function in vitro, and ectopic expression of MTS2inhibits cell growth in vivo. The above results raise the possibilitythat despite the biochemical similarity between MTS2 and MTS1, the twoproteins have significantly different functions in vivo. Two features ofMTS2 suggest that this may be so: i) MTS2, not MTS1, is induced by TGFβ(Hannon and Beach, 1994) and ii) unlike MTS1, MTS2 transcription appearsto be independent of Rb. It is possible that MTS2 is not involved intumorigenesis at all. Alternatively, MTS2 may participate in a pathwayof tumor suppression distinct from the pathway involving MTS1. Theelements of this pathway are not known, but it is conceivable that someof these elements may mutate at much higher frequencies than MTS2 insomatic tissue. In this view, the lack of somatic mutation of MTS2 in noway precludes an important role in tumor suppression. As noted above,ectopic expression of MTS2 inhibits cell growth, a role consistent withMTS2 being a tumor suppressor. The constant level of MTS2 expressionduring the cell cycle, and its induction by TGFβ, suggest a role forMTS2 in G1 arrest and not necessarily in regulating the timing of eventsin the cell cycle itself In contrast, the regulation of MTS1 expressionby Rb indicates that MTS1 may have a role in a cell cycle oscillator. Itwill be important to test the function of MTS2 as a growth controlmolecule in vivo, and to dissect the pathway(s) within which MTS2functions.

EXAMPLE 15 Two Step Assay to Detect the Presence of MTS in a Sample

Patient sample is processed according to the method disclosed byAntonarakis, et al. (1985), separated through a 1% agarose gel andtransferred to nylon membrane for Southern blot analysis. Membranes areUV cross linked at 150 mJ using a GS Gene Linker (Bio-Rad). MTS probecorresponding to nucleotide positions 448-498 of SEQ ID NO:4 issubcloned into pTZ18U. The phagemids are transformed into E. coli MV1190infected with M13KO7 helper phage (Bio-Rad, Richmond, Calif.). Singlestranded DNA is isolated according to standard procedures (see Sambrook,et al., 1989).

Blots are prehybridized for 15-30 min. at 65° C. in 7% sodium dodecylsulfate (SDS) in 0.5 M NaPO₄. The methods follow those described byNguyen, et al., 1992. The blots are hybridized overnight at 65° C. in 7%SDS, 0.5M NaPO₄ with 25-50 ng/ml single stranded probe DNA.Post-hybridization washes consist of two 30 min washes in 5% SDS, 40 mMNaPO₄ at 65° C., followed by two 30-min washes in 1% SDS, 40 mM NaPO₄ at65° C.

Next the blots are rinsed with phosphate buffered saline (pH 6.8) for 5min at room temperature and incubated with 0.2% casein in PBS for 30-60min. at room temperature and rinsed in PBS for 5 min. The blots are thenpreincubated for 5-10 minutes in a shaking water bath at 45° C. withhybridization buffer consisting of 6M urea, 0.3 M NaCl, and 5×Denhardt's solution (see Sambrook, et al., 1989). The buffer is removedand replaced with 50-75 μl/cm² fresh hybridization buffer plus 2.5 nM ofthe covalently cross-linked oligonucleotide-alkaline phosphataseconjugate with the nucleotide sequence complementary to the universalprimer site (UP-AP, Bio-Rad). The blots are hybridized for 20-30 min at45° C. and post hybridization washes are incubated at 45° C. as two 10min washes in 6M urea, 1× standard saline citrate (SSC), 0.1% SDS andone 10 min wash in 1× SSC, 0.1% Triton®X-100. The blots are rinsed for10 min. at room temp. with 1× SSC.

Blots are incubated for 10 min at room temperature with shaking in thesubstrate buffer consisting of 0.1 M diethanolamine, 1 mM MgCl₂, 0.02%sodium azide, pH 10.0. Individual blots are placed in heat sealable bagswith substrate buffer and 0.2 mM AMPPD(3-(2′-spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)phenyl-1,2-dioxetane,disodium salt, Bio-Rad). After a 20 min. incubation at room temperaturewith shaking, the excess AMPPD solution is removed. The blot is exposedto X-ray film overnight. Positive bands indicate the presence of MTS.

EXAMPLE 16 Generation of Polyclonal Antibody Against MTS

Segments of MTS coding sequence were expressed as fusion protein in E.coli. The overexpressed protein was purified by gel elution and used toimmunize rabbits and mice using a procedure similar to the one describedby Harlow and Lane, 1988. This procedure has been shown to generate Absagainst various other proteins (for example, see Kraemer, et al., 1993).

Briefly, a stretch of MTS coding sequence was cloned as a fusion proteinin plasmid PET5A (Novagen, Inc., Madison, Wis.). The MTS incorporatedsequence includes the amino acids corresponding to 448-498 of SEQ IDNO:4. After induction with IPTG, the overexpression of a fusion proteinwith the expected molecular weight was verified by SDS/PAGE. Fusionprotein was purified from the gel by electroelution. The identificationof the protein as the MTS fusion product was verified by proteinsequencing at the N-terminus. Next, the purified protein was used asimmunogen in rabbits. Rabbits were immunized with 100 μg of the proteinin complete Freund's adjuvant and boosted twice in 3 week intervals,first with 100 μg of immunogen in incomplete Freund's adjuvant followedby 100 μg of immunogen in PBS. Antibody containing serum is collectedtwo weeks thereafter.

This procedure is repeated to generate antibodies against the mutantforms of the MTS gene. These antibodies, in conjunction with antibodiesto wild type MTS, are used to detect the presence and the relative levelof the mutant forms in various tissues and biological fluids.

EXAMPLE 17 Generation of Monoclonal Antibodies Specific for MTS

Monoclonal antibodies are generated according to the following protocol.Mice are immunized with immunogen comprising intact MTS or MTS peptides(wild type or mutant) conjugated to keyhole limpet hemocyanin usingglutaraldehyde or EDC as is well known.

The immunogen is mixed with an adjuvant. Each mouse receives fourinjections of 10 to 100 μg of immunogen and after the fourth injectionblood samples are taken from the mice to determine if the serum containsantibody to the immunogen. Serum titer is deterimined by ELISA or RIA.Mice with sera indicating the presence of antibody to the immunogen areselected for hybridoma production.

Spleens are removed from immune mice and a single cell suspension isprepared (see Harlow and Lane, 1988). Cell fusions are performedessentially as described by Kohler and Milstein, 1975. Briefly, P3.65.3myeloma cells (American Type Culture Collection, Rockville, Md.) arefused with immune spleen cells using polyethylene glycol as described byHarlow and Lane, 1988. Cells are plated at a density of 2×10⁵ cells/wellin 96 well tissue culture plates. Individual wells are examined forgrowth and the supernatants of wells with growth are tested for thepresence of MTS specific antibodies by ELISA or RIA using wild type ormutant MTS target protein. Cells in positive wells are expanded andsubcloned to establish and confirm monoclonality.

Clones with the desired specificities are expanded and grown as ascitesin mice or in a hollow fiber system to produce sufficient quantities ofantibody for characterization and assay development.

EXAMPLE 18 Sandwich Assay for MTS

Monoclonal antibody is attached to a solid surface such as a plate,tube, bead, or particle. Preferably, the antibody is attached to thewell surface of a 96-well ELISA plate. 100 μl sample (e.g., serum,urine, tissue cytosol) containing the MTS peptide/protein (wild-type ormutants) is added to the solid phase antibody. The sample is incubatedfor 2 hrs at room temperature. Next the sample fluid is decanted, andthe solid phase is washed with buffer to remove unbound material. 100 μlof a second monoclonal antibody (to a different determinant on the MTSpeptide/protein) is added to the solid phase. This antibody is labeledwith a detector molecule (e.g., 125-I, enzyme, fluorophore, or achromophore) and the solid phase with the second antibody is incubatedfor two hrs at room temperature. The second antibody is decanted and thesolid phase is washed with buffer to remove unbound material.

The amount of bound label, which is proportional to the amount of MTSpeptide/protein present in the sample, is quantitated. Separate assaysare performed using monoclonal antibodies which are specific for thewild-type MTS as well as monoclonal antibodies specific for each of themutations identified in MTS.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

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36 471 base pairs nucleic acid double linear cDNA NO NO Homo sapiens CDS1..471 1 ATG GAG CCG GCG GCG GGG AGC AGC ATG GAG CCT TCG GCT GAC TGG CTG48 Met Glu Pro Ala Ala Gly Ser Ser Met Glu Pro Ser Ala Asp Trp Leu 1 510 15 GCC ACG GCC GCG GCC CGG GGT CGG GTA GAG GAG GTG CGG GCG CTG CTG 96Ala Thr Ala Ala Ala Arg Gly Arg Val Glu Glu Val Arg Ala Leu Leu 20 25 30GAG GCG GGG GCG CTG CCC AAC GCA CCG AAT AGT TAC GGT CGG AGG CCG 144 GluAla Gly Ala Leu Pro Asn Ala Pro Asn Ser Tyr Gly Arg Arg Pro 35 40 45 ATCCAG GTC ATG ATG ATG GGC AGC GCC CGA GTG GCG GAG CTG CTG CTG 192 Ile GlnVal Met Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu Leu 50 55 60 CTC CACGGC GCG GAG CCC AAC TGC GCC GAC CCC GCC ACT CTC ACC CGA 240 Leu His GlyAla Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu Thr Arg 65 70 75 80 CCC GTGCAC GAC GCT GCC CGG GAG GGC TTC CTG GAC ACG CTG GTG GTG 288 Pro Val HisAsp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val Val 85 90 95 CTG CAC CGGGCC GGG GCG CGG CTG GAC GTG CGC GAT GCC TGG GGC CGT 336 Leu His Arg AlaGly Ala Arg Leu Asp Val Arg Asp Ala Trp Gly Arg 100 105 110 CTG CCC GTGGAC CTG GCT GAG GAG CTG GGC CAT CGC GAT GTC GCA CGG 384 Leu Pro Val AspLeu Ala Glu Glu Leu Gly His Arg Asp Val Ala Arg 115 120 125 TAC CTG CGCGCG GCT GCG GGG GGC ACC AGA GGC AGT AAC CAT GCC CGC 432 Tyr Leu Arg AlaAla Ala Gly Gly Thr Arg Gly Ser Asn His Ala Arg 130 135 140 ATA GAT GCCGCG GAA GGT CCC TCA GAC ATC CCC GAT TGA 471 Ile Asp Ala Ala Glu Gly ProSer Asp Ile Pro Asp * 145 150 155 156 amino acids amino acid linearprotein unknown 2 Met Glu Pro Ala Ala Gly Ser Ser Met Glu Pro Ser AlaAsp Trp Leu 1 5 10 15 Ala Thr Ala Ala Ala Arg Gly Arg Val Glu Glu ValArg Ala Leu Leu 20 25 30 Glu Ala Gly Ala Leu Pro Asn Ala Pro Asn Ser TyrGly Arg Arg Pro 35 40 45 Ile Gln Val Met Met Met Gly Ser Ala Arg Val AlaGlu Leu Leu Leu 50 55 60 Leu His Gly Ala Glu Pro Asn Cys Ala Asp Pro AlaThr Leu Thr Arg 65 70 75 80 Pro Val His Asp Ala Ala Arg Glu Gly Phe LeuAsp Thr Leu Val Val 85 90 95 Leu His Arg Ala Gly Ala Arg Leu Asp Val ArgAsp Ala Trp Gly Arg 100 105 110 Leu Pro Val Asp Leu Ala Glu Glu Leu GlyHis Arg Asp Val Ala Arg 115 120 125 Tyr Leu Arg Ala Ala Ala Gly Gly ThrArg Gly Ser Asn His Ala Arg 130 135 140 Ile Asp Ala Ala Glu Gly Pro SerAsp Ile Pro Asp 145 150 155 1149 base pairs nucleic acid double linearDNA (genomic) NO NO Homo sapiens 5′UTR 1..866 CDS 867..1016 intron1017..1149 3 TCCCCCGCCC GTWTTAAWTA AACCTCATCT TTCCAGAGTC TGTTCTTATACCAGGAAATG 60 TACACGTCTG AGAAACCCTT GCCCCAGACA GTCGTTTTAC ACGCAGGAGGGGAAGGGGAG 120 GGGAAGGAGA GAGCAGTCCT TTTCTCCAAA AGGAATCCTT NGAACTAGGGTTTCTGACTT 180 AGTGAACCCC GCGYTCCTGA AAATCAWGGG TTGAGGGGGT AGGGGGACACTTYCCTAGTC 240 GYACAGSTKA TTTCGMTYCT CGGTGGGGCT CTCACAMCTA GGAAAGAATWGTTTTGCTTT 300 TTCTTATGAT TAAAAGAAGA AGCCATACTT TTCCCTATGA CACCAAACACCCCGATTCAA 360 TTTGGCAGTT AGGAAGGTTG TATCGCGGAG GAAGGAAACG GGGCGGGGGCGGATTTCTTT 420 TTTAACAGAG TGAACGCACT CAAACACGCC TTTGCTGGCA GGCGGGGGGAGCGCGGCTGG 480 GAGCAGGGGA GGCCGGAGGG CGGTGTGGGG GGCAGGTGGG GAGGAGCCCAGTCCTCCTTC 540 CTTGCCAACG CTGGCTCTGG CGAGGGCTGC TTYCGGCTGG TGCCCCCGGGGGAGACCCAA 600 CCTGGGGCGA CTTCAGGGGT GCCACATTCG CTAAGTGCTC GGAGTTAATAGCACCTCCTC 660 CGAGCACTCG CTCACAGCGT CCCCTTGCCT GGAAAGATAC CGCGGTCCCTCCAGAGGATT 720 TGAGGGACAG GGTCGGAGGG GGCTCTTCCG CCAGCACCGG AGGAAGAAAGAGGAGGGGCT 780 GGCTGGTCAC CAGAGGGTGG GGCGGACCGC GTGCGCTCGG CGGCTGCGGAGAGGGGGAGA 840 GCAGGCAGCG GGCGGCGGGG AGCAGCATGG AGCCGGCGGC GGGGAGCAGCATGGAGCCTT 900 CGGCTGACTG GCTGGCCACG GCCGCGGCCC GGGGTCGGGT AGAGGAGGTGCGGGCGCTGC 960 TGGAGGCGGG GGCGCTGCCC AACGCACCGA ATAGTTACGG TCGGAGGCCGATCCAGGTGG 1020 GTAGAGGGTC TGCAGCGGGA GCAGGGGATG GCGGGCGACT CTGGAGGACGAAGTTTGCAG 1080 GGGAATTGGA ATCAGGTAGC GCTTCGATTC TCCGGAAAAA GGGGAGGCTTCCTGGGGAGT 1140 TTTCAGAAC 1149 1187 base pairs nucleic acid doublelinear DNA (genomic) NO NO Homo sapiens intron 1..191 exon 192..498intron 499..1187 4 GAATTCATTG TGTACTGAAG AATGGATAGA GAACTCAAGAAGGAAATTGG AAACTGGAAG 60 CAAATGTAGG GGTAATTAGA CACCTGGGGC TTGTGTGGGGGTCTGCTTGG CGGTGAGGGG 120 GCTCTACACA AGCTTCCTTT CCGTCATGCC GGCCCCCACCCTGGCTCTGA CCATTCTGTT 180 CTCTCTGGCA GGTCATGATG ATGGGCAGCG CCCGAGTGGCGGAGCTGCTG CTGCTCCACG 240 GCGCGGAGCC CAACTGCGCC GACCCCGCCA CTCTCACCCGACCCGTGCAC GACGCTGCCC 300 GGGAGGGCTT CCTGGACACG CTGGTGGTGC TGCACCGGGCCGGGGCGCGG CTGGACGTGC 360 GCGATGCCTG GGGCCGTCTG CCCGTGGACC TGGCTGAGGAGCTGGGCCAT CGCGATGTCG 420 CACGGTACCT GCGCGCGGCT GCGGGGGGCA CCAGAGGCAGTAACCATGCC CGCATAGATG 480 CCGCGGAAGG TCCCTCAGGT GAGGACTGAT GATCTGAGAATTTGTACYCT GAGAGCTTCC 540 AAAGCTCAGA GCATTCATTT TCCAGCACAG AAAGTTCAGCCCGGGAGACC AGTCTCCGGT 600 CTTGCGCTCA GCTCACGCGC CAATGCGGTG GGACGGCCTGAGTCTCCCTA TGCGCCCTGC 660 CSCGCACAGC GCGGCAAATG GGAAATAATC CCGAAATGGACTTGCGCACG TGAAAGCCCA 720 TTTTGTACGT TATACTTCCC AAAGCATACC ACCACCCAAACACCTACCCT CTGCTAGTTC 780 AAGGCCTAGA CTGCGGAGCA ATGAAGACTC AAGAGGCTAGAGGTCTAGTG CCCCCTCTTC 840 CTCCAAACTA GGGCCAGTTG CATCSACTTA CCAGGTCTGTTTCCTCATTT GCATACCAAG 900 CTGGCTGGAC CAACCTCAGG ATTTCCAAAC CCAATTGTGCGTGGCATCAT CTGGAGATCT 960 CTCGATCTCG GCTCTTCTGC ACAACTCAAC TAATCTGACCCTCCTCAGCT AATCTGACCC 1020 TCCGCTTTAT GCGGTAGAGT TTTCCAGAGC TGCCCCAGGGGGTTCTGGGG ACATCAGGAC 1080 CAAGACTTCG CTGACCCTGG CAGTCTGTGC ACCGGAGTTGGCTCCTTTCC CTCTTAAACT 1140 TGTGCAAGAG ATCCCTATAG TGAGTCGTAT TATNCGGCCGCGAATTC 1187 1244 base pairs nucleic acid double linear DNA (genomic) NONO Homo sapiens intron 1..273 misc_RNA 274..529 /note= “Corresponds toexon of SEQ ID NO4” 5 GATCATCACT TTACCATCAA CTTTCTTGTC TCTGAACGTTTAGAGAATAA AATGGCATTT 60 AATTGGTVCT GAGTWTAACC TGAAGGTGGG GTGGGAAAGTGGWTTGCATC AGCAADTGAA 120 GAAACACCAG ACATCAGAGA CCTGAACACC TCTGCACTGGGTGAAAACTT GGCAATTAGG 180 TGTTTCTTTA AATGGCTCCA CCTGCCTTGC CCCGGCCGGCATCTCCCATA CCTGCCCCCA 240 CCCTGGCTCT GACCACTCTG CTCTCTCTGG CAGGTCATGATGATGGGCAG CGCCCGCGTG 300 GCGGAGCTGC TGCTGCTCCA CGGCGCGGAG CCCAACTGCGCAGACCCTGC CACTCTCACC 360 CGACCGGTGC ATGATGCTGC CCGGGAGGGC TTCCTGGACACGCTGGTGGT GCTGCACCGG 420 GCCGGGGCGC GGCTGGACGT GCGCGATGCC TGGGGTCGTCTGCCCGTGGA CTTGGCCGAG 480 GAGCGGGGCC ACCGCGACGT TGCAGGGTAC CTGCGCACAGCCACGGGGGA CTGACGCCAG 540 GTTCCCCAGC CGCCCACAAC GACTTTATTT TCTTACCCAATTTCCCACCC CCACCCACCT 600 AATTCGATGA AGGCTGCCAA CGGGGAGCGG CGGAAAGCCTGTAAGCCTGC AAGCCTGTCT 660 GAGACTCACA GGAAGGAGGA GCCGACCGGG AATAACCTTCCATACATTTT TTTCTTTGTC 720 TTATCTGGCC CTCGACACTC ACCATGAAGC GAAACACAGAGAAGCGGATT TCCAGGGATA 780 TTTAGGAGTG TGTGACATTC CAGGGGTCGT TTGNTTTTCAGGGTTTTCTG AGGGAAAGTG 840 CATATGAAAT CCTTGACTGG ACCTGGTGGC TACGAATCTTCCCGATGGAT GAATCTCCCA 900 CTCCAGCGCT GAGTGGGAGA AGGCAGTGAT TAGCACTTGGGTGACGGCAG TCGATGCGTT 960 CACTCCAATG TCTGCTGAGG AGTTATGGTG AACCCACAACTTAGGCCCTA GCGGCAGAAA 1020 GGAAAACCTG AAGACTGAGG ACAAAGTGGA GGAGGGCCGAGGTGGGCTTC AGTATGTCCC 1080 CNNCGGCGCT TTAGTTTGAG CGCATGGCAA GTCACATGCGTAAACGACAC TCTCTGGAAG 1140 CCCTGGAGAC CCTCGCCCAA CTCCACCAGA TAGCAGAGGGGTAAGAGAGG ATGTGCAAGC 1200 GACGACAGAT GCTAAAATCC CTGGATCACG ACGCTGCAGAGCAC 1244 19 base pairs nucleic acid single linear DNA (genomic) NO NOHomo sapiens 6 CAGCACCGGA GGAAGAAAG 19 20 base pairs nucleic acid singlelinear DNA (genomic) NO YES Homo sapiens 7 GCGCTACCTG ATTCCAATTC 20 20base pairs nucleic acid single linear DNA (genomic) NO NO Homo sapiens 8GGAAATTGGA AACTGGAAGC 20 19 base pairs nucleic acid single linear DNA(genomic) NO YES Homo sapiens 9 TCTGAGCTTT GGAAGCTCT 19 21 base pairsnucleic acid single linear DNA (genomic) NO NO Homo sapiens 10GATCATCACT TTACCATCAA C 21 19 base pairs nucleic acid single linear DNA(genomic) NO YES Homo sapiens 11 GGGTGGGAAA TTGGGTAAG 19 20 base pairsnucleic acid single linear DNA (genomic) NO NO Homo sapiens 12TGAGTTTAAC CTGAAGGTGG 20 1131 base pairs nucleic acid double linear cDNANO NO Homo sapiens CDS 338..655 13 CGCGCCTGCG GGGCGGAGAT GGGCAGGGGGCGGTGCGTGG GTCCCAGTCT GCAGTTAAGG 60 GGGCAGGAGT GGCGCTGCTC ACCTCTGGTGCCAAAGGGCG GCGCAGCGGC TGCCGAGCTC 120 GGCCCTGGAG GCGGCGAGAA CATGGTGCGCAGGTTCATGG TGACCCTCCG GATTCGGCGC 180 GCGTGCGGAC CGCCGCGAGT GAGGGTTTTCGTGGTTCACA TCCCGCGGCT CACGGGGGAG 240 TGGGCAGCAC CAGGGGCGCC CGCCGCTGTGGCCCTCGTGC TGATGCTACT GAGGAGCCAG 300 CGTCTAGGGC AGCAGCCGCT TCCTAGAAGACCAGGTC ATG ATG ATG GGC AGC GCC 355 Met Met Met Gly Ser Ala 160 CGA GTGGCG GAG CTG CTG CTG CTC CAC GGC GCG GAG CCC AAC TGC GCC 403 Arg Val AlaGlu Leu Leu Leu Leu His Gly Ala Glu Pro Asn Cys Ala 165 170 175 GAC CCCGCC ACT CTC ACC CGA CCC GTG CAC GAC GCT GCC CGG GAG GGC 451 Asp Pro AlaThr Leu Thr Arg Pro Val His Asp Ala Ala Arg Glu Gly 180 185 190 195 TTCCTG GAC ACG CTG GTG GTG CTG CAC CGG GCC GGG GCG CGG CTG GAC 499 Phe LeuAsp Thr Leu Val Val Leu His Arg Ala Gly Ala Arg Leu Asp 200 205 210 GTGCGC GAT GCC TGG GGC CGT CTG CCC GTG GAC CTG GCT GAG GAG CTG 547 Val ArgAsp Ala Trp Gly Arg Leu Pro Val Asp Leu Ala Glu Glu Leu 215 220 225 GGCCAT CGC GAT GTC GCA CGG TAC CTG CGC GCG GCT GCG GGG GGC ACC 595 Gly HisArg Asp Val Ala Arg Tyr Leu Arg Ala Ala Ala Gly Gly Thr 230 235 240 AGAGGC AGT AAC CAT GCC CGC ATA GAT GCC GCG GAA GGT CCC TCA GAC 643 Arg GlySer Asn His Ala Arg Ile Asp Ala Ala Glu Gly Pro Ser Asp 245 250 255 ATCCCC GAT TGA AAGAACCAGA GAGGCTCTGA GAAACCTCGG GAAACTTAGA 695 Ile ProAsp * 260 TCATCAGTCA CCGAAGGTCC TACAGGGCCA CAACTGCCCC CGCCACAACCCACCCCGCTT 755 TCGTAGTTTT CATTTAGAAA ATAGAGCTTT TAAAAATGTC CTGCCTTTTAACGTAGATAT 815 AAGCCTTCCC CCACTACCGT AAATGTCCAT TTATATCATT TTTTATATATTCTTATAAAA 875 ATGTAAAAAA GAAAAACACC GCTTCTGCCT TTTCACTGTG TTGGAGTTTTCTGGAGTGAG 935 CACTCACGCC CTAAGCGCAC ATTCATGTGG GCATTTCTTG CGAGCCTCGCAGCCTCCGGA 995 AGCTGTCGAC TTCATGACAA GCATTTTGTG AACTAGGGAA GCTCAGGGGGGTTACTGGCT 1055 TCTCTTGAGT CACACTGCTA GCAAATGGCA GAACCAAAGC TCAAATAAAAATAAAATTAT 1115 TTTCATTCAT TCACTC 1131 105 amino acids amino acid linearprotein unknown 14 Met Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu LeuLeu His Gly 1 5 10 15 Ala Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu ThrArg Pro Val His 20 25 30 Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu ValVal Leu His Arg 35 40 45 Ala Gly Ala Arg Leu Asp Val Arg Asp Ala Trp GlyArg Leu Pro Val 50 55 60 Asp Leu Ala Glu Glu Leu Gly His Arg Asp Val AlaArg Tyr Leu Arg 65 70 75 80 Ala Ala Ala Gly Gly Thr Arg Gly Ser Asn HisAla Arg Ile Asp Ala 85 90 95 Ala Glu Gly Pro Ser Asp Ile Pro Asp 100 105751 base pairs nucleic acid double linear cDNA NO NO Homo sapiens CDS335..751 15 CGGGCAGTGA GGACTCCGCG ACGCGTCCGC ACCCTGCGGC CAGAGCGGCTTTGAGCTCGG 60 CTGCGTCCGC GCTAGGCGCT TTTTCCCAGA AGCAATCCAG GCGCGCCCGCTGGTTCTTGA 120 GCGCCAGGAA AAGCCCGGAG CTAACGACCG GCCGCTCGGC CACTGCACGGGGCCCCAAGC 180 CGCAGAAGGA CGACGGGAGG GTAATGAAGC TGAGCCCAGG TCTCCTAGGAAGGAGAGAGT 240 GCGCCGGAGC AGCGTGGGAA AGAAGGGAAG AGTGTCGTTA AGTTTACGGCCAACGGTGGA 300 TTATCCGGGC CGCTGCGCGT CTGGGGGCTG CGGA ATG CGC GAG GAG AACAAG 352 Met Arg Glu Glu Asn Lys 110 GGC ATG CCC AGT GGG GGC GGC AGC GATGAG GGT CTG GCC AGC GCC GCG 400 Gly Met Pro Ser Gly Gly Gly Ser Asp GluGly Leu Ala Ser Ala Ala 115 120 125 GCG CGG GGA CTA GTG GAG AAG GTG CGACAG CTC CTG GAA GCC GGC GCG 448 Ala Arg Gly Leu Val Glu Lys Val Arg GlnLeu Leu Glu Ala Gly Ala 130 135 140 GAT CCC AAC GGA GTC AAC CGT TTC GGGAGG CGC GCG ATC CAG GTC ATG 496 Asp Pro Asn Gly Val Asn Arg Phe Gly ArgArg Ala Ile Gln Val Met 145 150 155 160 ATG ATG GGC AGC GCC CGC GTG GCGGAG CTG CTG CTG CTC CAC GGC GCG 544 Met Met Gly Ser Ala Arg Val Ala GluLeu Leu Leu Leu His Gly Ala 165 170 175 GAG CCC AAC TGC GCA GAC CCT GCCACT CTC ACC CGA CCG GTG CAT GAT 592 Glu Pro Asn Cys Ala Asp Pro Ala ThrLeu Thr Arg Pro Val His Asp 180 185 190 GCT GCC CGG GAG GGC TTC CTG GACACG CTG GTG GTG CTG CAC CGG GCC 640 Ala Ala Arg Glu Gly Phe Leu Asp ThrLeu Val Val Leu His Arg Ala 195 200 205 GGG GCG CGG CTG GAC GTG CGC GATGCC TGG GGT CGT CTG CCC GTG GAC 688 Gly Ala Arg Leu Asp Val Arg Asp AlaTrp Gly Arg Leu Pro Val Asp 210 215 220 TTG GCC GAG GAG CGG GGC CAC CGCGAC GTT GCA GGG TAC CTG CGC ACA 736 Leu Ala Glu Glu Arg Gly His Arg AspVal Ala Gly Tyr Leu Arg Thr 225 230 235 240 GCC ACG GGG GAC TGA 751 AlaThr Gly Asp * 245 138 amino acids amino acid linear protein unknown 16Met Arg Glu Glu Asn Lys Gly Met Pro Ser Gly Gly Gly Ser Asp Glu 1 5 1015 Gly Leu Ala Ser Ala Ala Ala Arg Gly Leu Val Glu Lys Val Arg Gln 20 2530 Leu Leu Glu Ala Gly Ala Asp Pro Asn Gly Val Asn Arg Phe Gly Arg 35 4045 Arg Ala Ile Gln Val Met Met Met Gly Ser Ala Arg Val Ala Glu Leu 50 5560 Leu Leu Leu His Gly Ala Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu 65 7075 80 Thr Arg Pro Val His Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu 8590 95 Val Val Leu His Arg Ala Gly Ala Arg Leu Asp Val Arg Asp Ala Trp100 105 110 Gly Arg Leu Pro Val Asp Leu Ala Glu Glu Arg Gly His Arg AspVal 115 120 125 Ala Gly Tyr Leu Arg Thr Ala Thr Gly Asp 130 135 57 basepairs nucleic acid single linear DNA (genomic) NO NO Homo sapiens 17AAAGGATCCA TTGCCACCAT GGAGCCGGCG GCGGGGAGCA GCATGGAGCC TTCGGCT 57 24base pairs nucleic acid single linear DNA (genomic) NO YES Homo sapiens18 TTTGAATTCA ATCGGGGATG TCTG 24 19 base pairs nucleic acid singlelinear DNA (genomic) NO NO Homo sapiens 19 AGGGAAGAGT GTCGTTAAG 19 20base pairs nucleic acid single linear DNA (genomic) NO YES Homo sapiens20 AGACTCCTGT ACAAATCTAC 20 16 base pairs nucleic acid single linear DNA(genomic) NO NO Homo sapiens 21 TGAGGGTCTG GCCAGC 16 17 base pairsnucleic acid single linear DNA (genomic) NO YES Homo sapiens 22AGCACCACCA GCGTGTC 17 16 base pairs nucleic acid single linear DNA(genomic) NO YES Homo sapiens 23 CGTGTCCAGG AAGCCC 16 144 base pairsnucleic acid double linear cDNA NO NO unknown 24 AATTCGGCAC GAGGCAGCATGGAGCCTTCG GCTGACTGGC TGGCCACGGC CGCGGCCCGG 60 GGTCGGGTAG AGGAGGTGCGGGCGCTGCTG GAGGCGGTGG CGCTGCCCAA CGCACCGAAT 120 AGTTACGGTC GGAGGCCGATCCAG 144 395 base pairs nucleic acid double linear cDNA NO NO unknown 25AAGAGAGGGT TTTCTTGGTA AAGTTCGTGC GATCCCGGAG ACCCAGGACA GCGTAGCTGC 60GCTCTGGCTT TCGTGAACAT GTTGTTGAGG CTAGAGAGGA TCTTGAGAAG AGGGCCGCAC 120CGGAATCCTG GACCAGGTGA TGATGATGGG CAACGTTCAC GTAGCAGCTC TTCTGCTCAA 180CTACGGTGCA GATTCGAACT GCGAGGACCC CACTACCTTC TCCCGCCCGG TGCACGACGC 240AGCGCGCGAA GGCTTCCTGG ACACGCTGGT GGTGCTGCAC GGGTCAGGGG CTCGGCTGGA 300TGTCCGCGAT GCCTGGGGTC GCCTCCCGCT CGACTTCGCC CAAGAGCGGG GACATCAAGA 360CATCGTGCGA TATTTGCGTT CCGCTGGGTG CTCTT 395 20 base pairs nucleic acidsingle linear cDNA NO unknown 26 CAACGCACCG AATAGTTACG 20 20 base pairsnucleic acid single linear cDNA NO unknown 27 TACTGAGGAG CCAGCGTCTA 2030 base pairs nucleic acid double linear other nucleic acid unknown 28TGAGTAGAAT TCTAACGGCC GTCATTGTTC 30 20 base pairs nucleic acid singlelinear cDNA NO unknown 29 AGCGTGTCCA GGAAGCCTTC 20 27 base pairs nucleicacid single linear other nucleic acid unknown 30 TGAGTAGAAT TCTAACGGCCGTCATTG 27 26 base pairs nucleic acid single linear cDNA NO unknown 31ACTGCGAGGA CCCCACTACC TTCTCC 26 20 base pairs nucleic acid single linearcDNA NO unknown 32 GAACGTTGCC CATCATCATC 20 15 base pairs nucleic acidsingle linear cDNA NO NO unknown 33 AGTCTGCAGT TAAGG 15 21 base pairsnucleic acid single linear cDNA NO YES unknown 34 GGCTAGAGGC GAATTATCTGT 21 21 base pairs nucleic acid single linear cDNA NO YES unknown 35CACCAAACAA AACAAGTGCC G 21 947 base pairs nucleic acid double linearcDNA NO NO Homo sapiens misc_feature 151 /note= “Splice site acceptor.”misc_feature 458 /note= “Splice site acceptor.” 36 ATGGAGCCGG CGGCGGGGAGCAGCATGGAG CCTTCGGCTG ACTGGCTGGC CACGGCCGCG 60 GCCCGGGGTC GGGTAGAGGAGGTGCGGGCG CTGCTGGAGG CGGGGGCGCT GCCCAACGCA 120 CCGAATAGTT ACGGTCGGAGGCCGATCCAG GTCATGATGA TGGGCAGCGC CCGAGTGGCG 180 GAGCTGCTGC TGCTCCACGGCGCGGAGCCC AACTGCGCCG ACCCCGCCAC TCTCACCCGA 240 CCCGTGCACG ACGCTGCCCGGGAGGGCTTC CTGGACACGC TGGTGGTGCT GCACCGGGCC 300 GGGGCGCGGC TGGACGTGCGCGATGCCTGG GGCCGTCTGC CCGTGGACCT GGCTGAGGAG 360 CTGGGCCATC GCGATGTCGCACGGTACCTG CGCGCGGCTG CGGGGGGCAC CAGAGGCAGT 420 AACCATGCCC GCATAGATGCCGCGGAAGGT CCCTCAGACA TCCCCGATTG AAAGAACCAG 480 AGAGGCTCTG AGAAACCTCGGGAAACTTAG ATCATCAGTC ACCGAAGGTC CTACAGGGCC 540 ACAACTGCCC CCGCCACAACCCACCCCGCT TTCGTAGTTT TCATTTAGAA AATAGAGCTT 600 TTAAAAATGT CCTGCCTTTTAACGTAGATA TAAGCCTTCC CCCACTACCG TAAATGTCCA 660 TTTATATCAT TTTTTATATATTCTTATAAA AATGTAAAAA AGAAAAACAC CGCTTCTGCC 720 TTTTCACTGT GTTGGAGTTTTCTGGAGTGA GCACTCACGC CCTAAGCGCA CATTCATGTG 780 GGCATTTCTT GCGAGCCTCGCAGCCTCCGG AAGCTGTCGA CTTCATGACA AGCATTTTGT 840 GAACTAGGGA AGCTCAGGGGGGTTACTGGC TTCTCTTGAG TCACACTGCT AGCAAATGGC 900 AGAACCAAAG CTCAAATAAAAATAAAATTA TTTTCATTCA TTCACTC 947

What is claimed is:
 1. An isolated DNA consisting of DNA having at least15 consecutive nucleotides of a DNA as represented by a nucleotidesequence selected from the group consisting of nucleotides 1-273 of SEQID NO:5, nucleotides 530-1244 of SEQ ID NO:5 and nucleotides 1-490 ofSEQ ID NO:15.
 2. A vector which comprises the isolated DNA of claim 1.3. A host cell transformed with the vector of claim
 2. 4. A replicativecloning vector which comprises the isolated DNA of claim 1 and areplicon operative in a host cell.
 5. Isolated host cells transformedwith the replicative cloning vector of claim
 4. 6. A pair of singlestranded DNA primers for determination of a nucleotide sequence of anMTS2 gene by a polymerase chain reaction, wherein the use of saidprimers in a polymerase chain reaction results in the synthesis of DNAhaving all or part of the sequence of the MTS2 gene, and further whereinsaid single-stranded primers are complementary to a DNA as representedby a nucleotide sequence selected from the group consisting ofnucleotides 1-273 of SEQ ID NO:5 or its complement, nucleotides 530-1244of SEQ ID NO:5 or its complement and nucleotides 1-490 of SEQ ID NO:15or its complement.
 7. The pair of primers of claim 6 wherein saidsequence of the MTS2 gene is set forth in SEQ ID NO:5.
 8. The pair ofprimers of claim 6 wherein said sequence of the MTS2 gene is set forthin SEQ ID NO:15.
 9. An oligonucleotide probe comprising 8 consecutivenucleotides complementary to a human wild-type MTS2 gene sequence, saidwild-type MTS2 gene sequence comprising a DNA as represented by anucleotide sequence selected from the group consisting of nucleotides1-273 of SEQ ID NO:5 or its complement, nucleotides 530-1244 of SEQ IDNO:5 or its complement and nucleotides 1-490 of SEQ ID NO:15 or itscomplement.
 10. A replicative cloning vector which comprises theoligonucleotide probe of claim 9 and a replicon operative in a hostcell.
 11. Host cells transformed with the replicative cloning vector ofclaim
 10. 12. A vector which comprises the oligonucleotide probe ofclaim
 8. 13. A host cell transformed with the vector of claim 12.